Kaarthik+R.

~**1.8** is generally accepted as “pure” for DNA ~**2.0-2.2**.
 * 260/280:** assess the purity of DNA and RNA
 * 260/230 Ratios:** measure of nucleic acid purity...often higher than the respective 260/280 values.
 * PfDXR extinction coefficient: ~39162.5 M-1 cm-1**
 * PfDXR molecular weight: 47206.6 Da**

HF9_180_Plates_1um3D_catnum.sdf HF9PlatesPlates5_9.sdf cb_306_3d.sdf NIH_ClinCol3Ded ChemBridge-3D Fragment-set_3D MayBridge50k_3D MW-set_3D
 * Screened Libraries (DONE)**

cb_306_3D.sdf ChemBridge-3D MayBridge50k_3D MW-set_3D HF9_Plates_1um3D_catnum.sdf
 * Useful Libraries** (Docking programs indicate compounds may be of use in these libraries)

chembIntd (Skip - paper indicates compounds do not target DXR) MAR3D7 (Skip - paper indicates compounds do not target DXR) Microsets
 * Screened Libraries (TO DO)**

PreFPLC PfDXR concentrated to 0.5 ml - takes about 50 min yield: 3.39 mg
 * January 21, 2013**

Used Umeda Buffers (all include 2 mM DTT - reducing agent) for protein purification Also learned what happens when the pH drops below 7 in protein solutions and when you fail to remove the sticker from the bottom the SDS Page Gel.


 * Week 13**
 * //Freebie from Journal Club Team B victory//**


 * Week 12**

//Enzymatic Assay not promising//
 * November 30, 2012**

Several enzyme assays (varying in room temp. vs 37 degrees Celsius and varying in duration from 5-15 min) were performed. Both snap frozen and -20 stocks of PfDXR were used. Absorbance of NADPH was measured at 340 nm. All trials revealed a constant gradient for NAPDH absorbance, indicating that NADPH was not being oxidized.

The obvious conclusion is that the PfDXR enzyme we synthesized is not functional. However, purification, characterization and concentration were all completed within a span of 12 hours, so the probability the enzyme denatured is low.

An alternative is to modify the assay parameters. Based on literature searches, the optimum temp. for PfDXR activity is 37 degrees Celsius. Most of the trials were done at room temp., which may explain why the absorbance was flat lined. For the two assays done at 37 degrees Celsius, one was done for 5 min. only while the other was "oversaturated" with enzyme - therefore, changing the optimal conc. of buffer and reagents. (NEED to verify the last sentence with data on computer in lab).

The remaining option is to redo the assay at 37 degrees Celsius with the following changes. 1. 5 min incubation with reagents and buffer at 37 degrees Celsius in PCR thermocycler. 2. Add PfDXR enzyme and obtain NADPH absorbance at 340 nm. 3. Incubate at 37 degrees Celsius for 10-20 min. 4. Read the absorbance value at 340 nm again.

If the above steps do not reveal a depletion of NADPH (i.e. 0 absorbance at 340 nm after 10-20 min), then all hope is lost - the enzyme is inactive.

//Prepared enzyme assay reagents//
 * November 28, 2012**

NADPH (cofactor) and DXP (substrate) were solubilized in 0.01 M NaOH and pure water, respectively to obtain 192 5 mM NADPH aliquots (5 enzyme assays each) and 175 12 mM DXP aliquots (1 enzyme assay each). Tris-HCl was diluted to 125 mM.

//Protein sonication (lysing), purification (extraction), characterization (verification), and concentration//
 * November 27, 2012**

PfDXR concentrated pre-FPLC (1 ml)

A = 0.235 e = 39162.5 M-1 cm-1 b = 1 cm

c = A/Eb = (0.235) / (39162.5 M-1 cm-1) * (1 cm) = 6 microMolar. 6 microMolar = (6 micro moles / 1 liter) * (47206.6 g/mole) = 0.283 mg/ml yield = 0.283 mg



PfDXR homodimer is present in fraction samples 25 through 29. Presence can be verified by molecular standards (77 kDa is the small bump near fraction samples 29 through 31). PfDXR monomer is approx. 47 kDa (dimer will be approx. 94 kDa).

Rescaled FPLC graph (same as above). Small bump at fractions 31 through 34 may indicate monomer - standard at fractions corresponding to 33 through 36 is approx. 33 kDa.

PfDXR concentrated post-FPLC in glycerol (0.5 ml)

A = 0.291 e = 39162.5 M-1 cm-1 b = 1 cm

c = A/Eb = (0.291) / (39162.5 M-1 cm-1) * (1 cm) = 7.43 microMolar. 7.43 microMolar = (7.43 micro moles / 1 liter) * (47206.6 g/mole) = 0.351 mg/ml yield: 0.1755 mg

PfDXR concentrated post-FPLC snap frozen (0.5 ml)

A = 0.294 e = 39162.5 M-1 cm-1 b = 1 cm

c = A/Eb = (0.294) / (39162.5 M-1 cm-1) * (1 cm) = 7.51 microMolar. 7.51 microMolar = (7.51 micro moles / 1 liter) * (47206.6 g/mole) = 0.354 mg/ml yield: 0.177 mg

total yield after FPLC: 0.354 mg

//IPTG induction, 20 hour overnight expression//
 * November 26, 2012**

112612 - ??. Dr B


 * Week 11**


 * November 21, 2012**

Ranking of Libraries by top scoring ligands (11/21/12) 1. ChemBridge-3D 2. HF9_180_Plates_1um3D_catnum 3. MayBridge50k_3D 4. MW-set_3D 5. cb_306_3d 6. NIH_ClinCol3Ded 7. Fragment-set_3D 8. HF9PlatesPlates5_9

Top Ten Ligand List did not change from Week 8 results. However, MayBridge50k_3D entered the top 20 list and MW-set_3D entered the top 30 list.

Top Compound No. 1 7576671 docked in active site of PfDXR 7576671 shown as sticks (Carbon purple). MG ion shown as sphere (Orange). Active site residues within 5 A shown as sticks (Carbon light blue). Hydrophobic residues shown as lines (Carbon green)

Top Compound No. 3 7969923 docked in active site of PfDXR

7969923 shown as sticks (Carbon purple). MG ion shown as sphere (Orange). Active site residues within 5 A shown as sticks (Carbon light blue). Hydrophobic residues shown as lines (Carbon green)

Top Compound No. 4 5583722 docked in active site of PfDXR 5583722 shown as sticks (Carbon purple). MG ion shown as sphere (Orange). Active site residues within 5 A shown as sticks (Carbon light blue). Hydrophobic residues shown as lines (Carbon green)

FOM docked in active site of PfDXR

FOM shown as sticks (Carbon purple). MG ion shown as sphere (Orange). Active site residues within 5 A shown as sticks (Carbon light blue). Hydrophobic residues shown as lines (Carbon green)


 * November 20, 2012**

We went ahead and transformed colonies 5 and 15 into BL21 (DE3) competent cells. Also, we started a 320mL culture of colony 5 for Midiprep. Will spin down and Midiprep tomorrow.

//DNA sequencing shows positive clones for submitted samples//
 * November 19, 2012**

Miniprepped plasmid samples from colonies 5,6,15,16, 21 & 22. A BLAST comparison indicated positive clones for every colony without mutations.


 * Week 10 -**
 * good - Dr. B 11/19/12**

//RE Digest with HincII enzyme to check for positive clones//
 * November 18, 2012**

Virtual Gel of PfDXRL75 - positive clones on gel image should have bands at sizes indicated above.



//Gel Image of RE Digest of PfDXR in pNIC-Bsa4// //with HincII for Colonies 1-8// Lane 1: Skip Lane 2: 1kb Ladder Lane 3: Colony 1 - not likely to be full insert Lane 4: Colony 2 - not likely to be full insert Lane 5: Colony 3 - not likely to be full insert Lane 6: Colony 4 - not likely to be full insert Lane 7: Colony 5 - positive clone (sequenced) Lane 8: Colony 6 - positive clone (sequenced) Lane 9: Colony 7 - likely a positive clone (did not sequence) Lane 10: Colony 8 - likely a positive clone (did not sequence)

//Gel Image of RE Digest of PfDXR in pNIC-Bsa4// //with HincII for Colonies 9-16// Lane 1: Skip Lane 2: 1kb Ladder Lane 3: Colony 9 - likely a positive clone (did not sequence) Lane 4: Colony 10 - likely a positive clone (did not sequence) Lane 5: Colony 11 - likely a positive clone (did not sequence) Lane 6: Colony 12 - likely a positive clone (did not sequence) Lane 7: Colony 13 - not likely to be full insert Lane 8: Colony 14 - not likely to be full insert Lane 9: Colony 15 - positive clone (sequenced) Lane 10: Colony 16 - positive clone (sequenced)

//Gel Image of RE Digest of PfDXR in pNIC-Bsa4// //with HincII for Colonies 17-24// Lane 1: Skip Lane 2: 1 kb Ladder Lane 3: Colony 17 - likely a positive clone (did not sequence) Lane 4: Colony 18 - likely a positive clone (did not sequence) Lane 5: Colony 19 - likely a positive clone (did not sequence) Lane 6: Colony 20 - not likely to be a positive clone Lane 7: Colony 21 - positive clone (sequenced) Lane 8: Colony 22 - positive clone (sequenced) Lane 9: Colony 23 - not likely to be a positive clone Lane 10: Colony 24 - not likely to be a positive clone

//Master Plate Protocol completed on 24 colonies//
 * November 17, 2012**

//Cut pNIC and Cloning and Transformation//
 * November 16, 2012**

//cut pNIC-Bsa4 at BsaI sites - combined two samples (starting conc. 82.4 ng/ul)//

//More PCR from plasmid// //combined 5 samples (20 cycles each)//
 * November 15, 2012**

//Back to cloning part 2//
 * November 14, 2012**

Transformation was not successful

//Back to cloning//
 * November 13, 2012**

We used the new L75 forward primer to do PCR.

Lane 1 & 5-10: Skip Lane 2: 1 kb DNA Ladder Lane 3: 10 ul PfDXRL75 (~1300 bp) - used 1 ng in 50 ul reaction volume, 20 cycles Lane 4: 10 ul PfDXRL75 (~1300 bp) - used 5 ng in 50 ul reaction volume, 20 cycles

PCR Clean Up was not as fruitful (21.9 ng/ul), but satisfactory


 * Week 9**

//PCR from primary mix- used wrong forward primer//
 * November 08, 2012**

supersec: 20 sec annealing kr2: 1 min annealing

Lane 3: 1A primary smear -primary mix made in June kr2 protocol Lane 4: 1B primary smear -primary mix made in July kr2 protocol

Lane 3: 1A primary smear - primary mix made in June supersec protocol Lane 4: 1B primary smear - primary mix made in July supersec protocol Lane 5: 2A secondary - kr2 protocol Lane 6: 2B secondary - kr2 protocol

secondary from 1B Lane 1&6: 100 bp Ladder Lane 2: 2B secondary - kr2 protocol Lane 3: 2B secondary - kr2 protocol Lane 4: 2B secondary - supersec protocol Lane 5: 2B secondary - supersec protocol

PCR^2 from 2B secondary -kr2 protocol

//Combined efforts no go - used wrong forward primer//
 * November 07, 2012**

Andrew, Alex and I gave it another attempt at PCR from the plasmid DNA.

Lane 1: 1kB DNA Ladder Lane 2-5: Alex's vain attempt using modified Aptamer PCR protocol on plasmid DNA (15 cycles) Lane 6: Kaarthik's vain attempt using custom protocol on plasmid DNA (30 cycles) Lane 7: Kaarthik's vain attempt using primary mix DNA with custom protocol. (30 cycles) Lane 8: Andrew's vain attempt using Aptamer protocol (no modifications) on plasmid DNA (15 cycles) Lane 9-12: Recheck of secondary PCR with new primers (20 cycles only).

Looks like we made a new DNA ladder!

We will remake the primary DNA and use the new forward to make secondary PCR DNA.

//PCR Party no go - used wrong forward primer//
 * November 04 and 05 and 06, 2012**

PCR on the plasmid DNA was attempted several times to no avail. I went back to the primary 1A mix (first mix made in the June 2012) and used it as template with the new forward primer. The secondary DNA output appeared to be the correct size in bp, but PCR^2 from this secondary resulted in smaller amplicon length.

After these successive failures, I will do a dpnI digest of the 17 ng/ul DNA and anneal/transform. The only downside is that the gene appears to be near 1500 bp (suggesting I used the wrong forward primer) but the gel can be deceiving.

Lane 1: 1kB DNA Ladder Lane 2-5: Secondary PCR^2 - wrong gene size Lane 6-12: Secondary PCR^2 - no DNA product Lane 1: 1kb DNA Ladder Lane 2: 1A Primer smear Lane 3-6: Secondary PCR from 1A primary Lane 7-10: Another attempt at DNA amplification from plasmid DNA

//PCR from plasmid DNA - used wrong forward primer//
 * November 02, 2012**

No PCR product - will probably need to adjust timing of denaturation and annealing steps -maybe try diluting stock plasmid directly (add 500 ul Tris HCl to original amount). -or do three fold dilution from 90 ng/ul to 45 ng/ul to 22.5 ng/ul to 11.25 ng/ul

//PCR from plasmid DNA. Incorrectly followed right protocol - used wrong forward primer//
 * November 01, 2012**

Lane 1 & 8-10: Skip Lane 2: 1 kb DNA Ladder Lane 3: PCR using overlap extension conditions - 84 ng of plasmid DNA (10 ul added to gel) Lane 4-7: PCR using plasmid DNA conditions

There was no product in lanes 4-7.

Mistakes made: 1. Used MgSO4 instead of MgCl2 2. Used PCR-Overlap thermocycling conditions instead of recommended protocol.

//PCR^2 from plasmid DNA. Followed wrong protocol - used wrong forward primer//
 * October 31, 2012**

DNA replication results were not promising. We used nearly 84 ng (the KOD info sheet recommends 1 ng only of plasmid DNA) so we need to modify the cycling conditions.


 * Week 8**

//Official No Wet Lab Week//

No significant work done in lab.

All of the jobs submitted to Maestro for 2D to 3D conversion of the chembIntd and MAR3D7 libraries failed to complete. One job succesfully ran but prematurely exited with a fatal message (FATAL m2io_get_number_blocks: error getting number of blocks in file) while the other 4 did not begin.

Virtual screening was completed on all of the proposed libraries. Some results follow.

Top Ten Ligands after 2 Runs of Screened Libraries 10/28/12

Ranking of Libraries by top scoring ligands 1. ChemBridge-3D 2. HF9_180_Plates_1um3D_catnum 3. cb_306_3d 4. NIH_ClinCol3Ded 5. Fragment-set_3D 6. HF9PlatesPlates5_9

Top Compound No. 2 RJC00044SC docked in active site of PfDXR

Validation docking was also completed (Autoscale = 2; comparable to above top ten) on known inhibitor Fosmidomycin (FOM) While FOM has a smaller Score than the top ten compounds, it has the second best S(hbond) and the best S(metal) score.

Original FOM docked in the active site of PfDXR Original FOM aligned with Validation Docking Pose No. 4 FOM The Validation indicates that Virtual Screening may provide reliable results for identifying inhibitors.


 * Week 7**

102112- Yeah - keep up the good work. Dr. B //Midiprepped PfDXR//
 * October 19, 2012**

Andrew spun down a culture of E. Coli with the recombinant DNA and the DNA was extracted with the Qiagen set.



//DNA Sequencing Results//
 * October 17, 2012**

Sample 2A5 has the full coding sequence in the pNIC-Bsa4 vector.

Sample 2A5. 77.1 ng/ul.


 * 2A5 Forward:**

>lcl|58321 Length=1890

Score = 1844 bits (998), Expect = 0.0 Identities = 1018/1031 (99%), Gaps = 4/1031 (0%) Strand=Plus/Plus

The Gaps included 2 deletions approximately 100 bp before the start codon which are likely false positives and 2 deletions around the 1050-1060 bp no. range and are false positives since these mutations were not present in the reverse sequence.

**2A5 Reverse**

>lcl|39031 Length=1890

Score = 1881 bits (1018), Expect = 0.0 Identities = 1030/1042 (99%), Gaps = 0/1042 (0%) Strand=Plus/Plus

There were no gaps in this read of the target coding sequence. There were several "N" - the actual bp at these locations was verified by reading the peaks displayed in the chromatogram and comparing base location to the forward read.

//DNA Sequencing Results//
 * October 16, 2012**

The predictions about which samples had the insert were wrong. Neglecting that, there were two positive clones in the sequencing results: 1A2 and 2A3. 1A3 had about 4 deletions and 2A3 had 1 deletion only. There may be more deletions (or other mutations) since we only sent the forward pLIC primer with the samples. Since we know the 2A samples are reliable, we will submit the following samples to sequencing:

2A3 with pLIC-rev, oligoprimer no. 1, oligoprimer no. 38 2A1 with pLIC-for 2A4 with pLIC-for 2A5 with pLIC-for and pLIC-rev

//DNA Sequencing Submission//
 * October 15, 2012**

The following samples were sent to sequencing: 1A3 1A2 1A5 1A6 1B2 2A3 2A5 2B6

All of them were submitted with the pLIC-for primer.

101612 - Kaarthik - ok good deal. Hopefully the DNA sequencing works. -- Dr. B

//Restriction Enzyme Digest of miniprep samples with HincII//
 * Week 6**
 * October 13, 2012**

The following restriction enzyme digest indicated the successful transformation of E. Coli DH5a with recombinant DNA pNIC-Bsa4 PfDXR construct, albeit only in a few cases. No DNA ladder was added to the agarose gel because 12 samples were added to each agarose gel (with 12 lanes).

HincII RE Digest of Samples from A & B plates containing 249.2 ng/ul insert.

Lane 1. 1A1 - likely insert Lane 2. 1A2 - unknown **10/16/12 INSERT: several deletions with pLIC-for** Lane 3. 1A3 - likely insert **10/16/12 NOT AN INSERT: tail end** Lane 4. 1A4 - no digest by HincII Lane 5. 1A5 - likely insert **10/16/12 NOT AN INSERT: tail end** Lane 6. 1A6 - likely insert **10/16/12 NOT AN INSERT: tail end** Lane 7. 1B1 - likely insert Lane 8. 1B2 - likely insert **10/16/12 NOT AN INSERT: tail end** Lane 9. 1B3 - no digest by HincII Lane 10. 1B4 - likely insert Lane 11. 1B5 - unknown Lane 12. 1B6 - no digest by HincII

The digest was repeated for the A & B plates containing the 339.1 ng/ul insert

HincII RE Digest of Samples from A & B plates containing 339.2 ng/ul insert

Lane 1. 2A1 - more than likely insert **10/17/12: INSERT: Several deletions in Insert** Lane 2. 2A2 - no digest by HincII Lane 3. 2A3 - more than likely insert **10/16/12: INSERT: only one deletion present with pLIC-for 10/17/12: Several deletions in Insert** Lane 4. 2A4 - more than likely insert **10/17/12: INSERT: Several deletions in Insert** Lane 5. 2A5 - confirmed insert **10/16/12: INSERT: MAGIC** Lane 6. 2A6 - no digest by HincII Lane 7. 2B1 - no DNA Lane 8. 2B2 - no digest by HincII Lane 9. 2B3 - no digest by HincII Lane 10. 2B4 - no DNA Lane 11. 2B5 - no digest by HincII Lane 12. 2B6 - more than likely insert **10/16/12: NOT AN INSERT: tail end**

For the above gel, we cannot conclude if the PfDXR gene is present because a successful cut of the recombinant DNA should produce 5 cuts, with 2 cuts visible and 3 cuts faded - see Sept. 14, 2012 or Sept. 24, 2012).

We made two mistakes in this trial. One, we should have added the DNA Ladder to make comparisons reliable and conclusions easier to validate. Second, we should have not run the gel for 45 minutes - 35 minutes at 110 V may be ideal for a RE Digest, especially since a digest with HincII will produce several DNA strands below 500 bp.

Another conclusion is to decrease the digest time to 1 hour since star activity is visible in both gels above. We will submit the following samples with the pNIC-for primer to DNA Sequencing:

1A3 1A2 1A5 1A6 1B2 2A3 2A5 2B6

//Miniprep RE Digest// Since there were 24 samples, only measurement (1A1) was uploaded.
 * October 12, 2012**

The 2B samples may be of low purity because we left ethanol solution in the sample tubes before proceeding to elution the next morning.

Nanodrop 1A1. Concentration 60.7 ng/ul 260/280 - 2.05 & 260/230 - 2.03

//Master Plate Protocol//
 * October 11, 2012**

Colonies grew overnight on the four plates. We chose 24 colonies (6 from each plate) to continue for miniprep.

//Cohesive End Generation, Annealing and Transformation//
 * October 10, 2012**

We ran a gel with 1 ul of the 340 ng/ul PfDXR cleaned up and 1 ul of the accepting vector cut with BsaI.

Gel indicating presence of 1.5kB PfDXR gene and cut pNIC-Bsa4

Lane 1: Skip Lane 2: 1 kB DNA Ladder Lane 3: PfDXR cleaned up (339.1 ng/ul) Lane 4: cut pNIC-Bsa4 Lane 5-10: Skip

We also did cohesive end generation with the new T4 polymerase, dGTP and dCTP (purchased in early October). We did the annealing and transformation step verbatim. Four plates were made (No. 1 contained the insert that originally came from the 249.2 concentrated stock and No. 2 contained the insert from the 339.1 concentrated stock) and one set of plates contained 2 ul of accepting vector + 4 ul of insert and the other set of plates contained 48 ul of accepting vector and 16 ul of insert.

//pNIC-Bsa4 Midiprep, pNIC-Bsa4 vector preparation//
 * October 09, 2012**

We midiprepped pNIC-Bsa4 according to the Qiagen protocol. We doubled the buffer volume (i.e. 6 ul to 12 ul) as was suggested in the booklet to increase DNA yield, but the result of this is uncertain because we ended with a low concentration of the vector - 51.5 ng/ul. The yield may also be low because we did not adhere strictly to time constraints (i.e. incubation times were longer).

Nanodrop of pNIC-Bsa4 eluted in 5 mM Tris HCl. Concentration was 51.5 ng/ul, 260/280 at 1.91, 260/230 at 2.29.

We also cut a 82 ng/ul pNIC-Bsa4 vector with BsaI. The results are promising. In the past, we let the RE Digest go for 2.5 hours, but we increased the time to 3 hours in this instance.

Nanodrop of pNIC-Bsa4 cut with BsaI eluted in 10 mM Tris HCl. Concentration was 71.7 ng/ul, 260/280 at 1.93, 260/230 at 2.30

//Secondary PCR, PCR^2//
 * October 08, 2012**

We successfully made high concentrations of the PfDXR encoding gene from PCR. We deviated from standard protocol in that we decreased the PCR^2 thermocycling to 25 cycles in the hopes of minimizing mutations.

Verification of Secondary PCR

Lane 1: Skip Lane 2: 1kB DNA Ladder (3 ul) Lane 3: Secondary PCR from primary mix C prepared in the beginning of July (5 ul) Lane 4-10: Skip

We used the KOD polymerase from 09/12/12 - we made sure to immediately transfer the final 50 ul sample to the thermocycler once the KOD was added. This was likely the critical step that we had failed to observe in last week's PCR attempts.

We then repeated the thermocycling with the secondary PCR template from above but did 25 cycles only.

Verification of PCR^2

Lane 1: Skip Lane 2: 1kB Ladder Lane 3: Secondary PCR^2 from primary C Lane 4: Secondary PCR^2 from primary C Lane 5: Secondary PCR^2 from primary C Lane 6: Secondary PCR^2 from primary C Lane 7-10: Skip

Since we hurried, the gel image is poor - we halted 20 minutes prematurely. Fortunately, the 1.5 kB band is visible in Lanes 3,4 and 5. The unevenness of the brightness in these lanes may be attributed to improper mixing of reagents prior to adding KOD polymerase to the master mix.

We then cleaned up the sample and eluted in 10 mM Tris-HCL. Nanodrop measurement of 2 ul of PfDXR eluted in 50 ul of 10 mM Tris HCl. Concentration at 249.2 ng/ul, 260/280 at 1.89 and 260/230 at 2.42.

The absorbance values are reasonable and the concentration broke a personal record set on July 09, 2012.

We checked the clean up below. Verification of PfDXR Gene after clean up

Lane 1: Skip Lane 2: 1kB DNA Ladder Lane 3: 2 ul of 249.2 ng/ul PfDXR cleaned up Lane 4-10: Skip

No tail end primers are visible - indicating minimal contamination.

We repeated the thermocycling to produce more PCR^2 to ensure we have enough for cloning. While the absorbance values are in range and the concentration is high, we will need to gel check the sample later to verify the presence of the PfDXR gene.

Nanodrop measurement of 2 ul of PfDXR (Sample No. 2) eluted in 50 ul 10 mM Tris HCL. Concentration at 339.1 ng/ul. 260/280 at 1.85, 260/230 at 2.27.

We will continue with cloning this week.


 * Week 5**


 * 100912 - look great. Keep on truckin' - DR. B**

//Verification of plates, PCR, Gel Check//
 * October 05, 2012**

The VDS Staff plates made in September were okay. At least 50 colonies grew on the plate containing SCP1 in pET. On our transformation attempt, the cohesive end generation may have failed - explaining the lack of colonies on the plates.

We also tried secondary PCR twice - once with the Q5 polymerase and the other with Phusion. Both attempts failed; it is possible that the PCR samples were left on ice too long after the polymerase was added.

Gel Check to verify the presence of DNA in samples We added 5 ul of sample to each lane

Lane 1: Skip Lane 2: 1kB DNA Ladder Lane 3: 1A - Primary prepared in Summer Lane 4: 1B - Primary prepared in Summer Lane 5: 1C - Primary prepared in Summer Lane 6: alpha - Primary prepared in Fall (09/06/12) Lane 7: gamma - Primary prepared in Fall (09/06/12) Lane 8: Secondary prepared in Summer Lane 9: gamma - Secondary prepared in Fall (from the Lane 7 sample)

Next week we will continue with making secondary PCR samples and attempt another transformation.

//Unsuccessful transformation of pNIC-Bsa4:PfalDXR in DH5-alpha.//
 * October 04, 2012**

No colonies grew on the four LB+Kan+5% Sucrose plates. Other colleagues did not have success on the same batch of plates so we transformed another kanamyacin resistant vector and YopH to determine the efficacy of the plates.

We also prepared another 20 plates with LB+Kan.+5% Sucrose.

//Annealing and Transformation Take 5, More PCR//
 * October 02, 2012**

The PCR Insert from October 01, 2012 was annealed with the cut pNIC-Bsa4, below. The RE Digest was done over 2.5 hours. Nanodrop measure of pNIC-Bsa4 digested with BsaI. Concentration 39.1 ng/ul, 260/280 at 2.01 and 260/230 at 2.16.

We also completed several PCR trials. The four PCR^2 samples were done with new dilutions of the forward and reverse primers.

Agarose Gel containing cut pNIC-Bsa4, secondary PCR samples and PCR^2 samples.

Lane 1: 1 kB DNA Ladder Lane 2: pNIC-Bsa4 Cut after PCR Clean Up Lane 3: Secondary gamma - original mix from primary 09/04/12 (3 ul) 20 cycles Lane 4: Secondary alpha - original mix from primary 09/04/12 (3 ul) 20 cycles Lane 5: Secondary gamma - original mix from primary 09/06/12 (3 ul) 25 cycles Lane 6: Secondary alpha - original mix from primary 09/06/12 (3 ul) 25 cycles Lane 7: Skip Lane 8: PCR^2 - original mix from secondary 07/18/12 (1 ul) 25 cycles Lane 9: PCR^2 - original mix from secondary 07/18/12 (1 ul) 25 cycles Lane 10: PCR^2 - original mix from secondary 07/18/12 (1 ul) 25 cycles Lane 11: PCR^2 - original mix from secondary 07/18/12 (1 ul) 25 cycles Lane 12 : Skip

We then cleaned up the PCR^2 samples from above. Unfortunately, the concentrations were too low for use and the samples were discarded. We performed two trials and deviated from the default protocol. Instead of combining all four PCR samples into one tube, we combined 2 samples into one tube and continued the clean up from there. As a result, only 100 ul of DNA sample was cleaned up in each trial.

Nanodrop measure of PfDXR; concentration 27.5 ng/ul, 260/280: 1.89, 260/230: 1.29 Nanodrop measure of PfDXR; concentration 29.7 ng/ul, 260/280: 1.99, 260/230: 1.72

//PCR No Fun//
 * October 01, 2012**

We did another PCR starting from the secondary mix. 4 copies were made, but all were done with 21 cycles only to minimize the possibility of mutations. Decreasing the cycles may have some tradeoffs, including decreased final product yield and increased contamination (more oligoprimers). Lane 1: Skip Lane 2: 1kb DNA Ladder Lane 3: Skip Lane 4: PCR^2 Sample 1 (3 ul) Lane 5: Skip Lane 6: PCR^2 Sample 2 (3 ul) Lane 7: Skip Lane 8: PCR^2 Sample 3 (3 ul) Lane 9: Skip Lane 10: PCR^2 Sample 4 (3 ul) Lane 11: Skip Lane 12: Skip

The difference in band intensity arises from unequal distribution of the master mix to the 4 aliquots. The gel also indicates the presence of a lot of smaller primers (tail end primers) in the PCR samples. This is likely a result of less cycling.

Interestingly, the concentration is comparatively high. The 260/230 absorbance ratio is less then preferred at 1.54

We ran 3 ul of the PCR cleanup Sample on a 1% agarose gel. Surprisingly, there does not seem to be a lot of contamination even though the nanodrop result indicated otherwise.

Lane 1: Skip Lane 2: 1 kB DNA Ladder Lane 3-9: Skip Lane 10: Pfal DXR Clean up (3 ul)

1000112 - Kaarthik - any updates? Dr. B


 * Week 4**

//DNA Sequencing Analysis for alpha 2, alpha 4, and alpha 12//
 * September 28, 2012**

From 16 colonies, 15 were successfully miniprepped. In the end, 3 colonies had the full coding DNA sequence, albeit with mutations. Most were deletions and are summarized below. The mutations were confirmed with DNA sequencing with five primers, pLIC-for, pLIC-rev, oligonucleotide 1, oligonucleotide 9, and oligonucleotide 38.


 * alpha 12**, at least 7 deletions at bp 423, 424, 524, 1371, 1372, 1374, 1376


 * alpha 2**, at least 7 deletions at bp 423, 424, 524, 1341, 1372, 1374, 1376


 * alpha 4**, at least 2 deletions confirmed at bp 404, 566, one point mutation at bp 898 G replaced for A, another 3 potential mutations at 406, 407, 408 (2 sequencing results indicated deletions at these sites, while one did not).

Since the mutations occur in multiple regions, it is improbable we can do site-directed mutagenesis on these samples. We will continue from secondary PCR but decrease cycling to 20 cycles. This may decrease the number of copies with mutations.

//Restriction enzyme Digest for 8 more colonies from alpha plate//
 * September 24, 2012**

We ran another digest on the 8 samples from Sept. 21, 2012. There was one positive clone, alpha-12, which was submitted for DNA Sequencing.

Lane 1: Skip Lane 2-8: alpha n (where n begins at 5 and ends at 11) Lane 9: alpha 12 pNIC-Bsa4 with Insert cut with HincII


 * Week 3**

//Blast Sequencing Results with custom oligo primers and growth of more colonies from the alpha plate.//
 * September 21 ,2012 - Kaarthik, looks good. Hopefully most of those indels won't turn out to be bad or else we can remove them with site-directed mutagenesis. -- Dr. B**

The results from sequencing were not positive. A2-for corroborated deletions at 578, 579 and the insertion at 679 while A3-rev corroborated deletions at 578, 579, 679, 1525, 1526, 1528, and 1530. Both A2-for and A2-rev revealed no deletion at 750, however. //NOTE: BP deletion sites are wrong. See Sept. 28 analysis for correct data. -- Sept. 28.//

The results from the A4 set were more promising but both A4-for and A4-rev indicated deletions at 558, and 720. At 560,561 and 562 there were no deletions and 372 was not sequenced by either DNA sample.

However, in anticipation of unpromising Sequencing results, another 8 colonies were selected from the alpha plate to grow overnight on 9/20. The nanodrop concentrations after miniprep are shown below.

The concentrations are much lower than shown in previous trials with other colonies. The yield may have decreased since the colonies grew over one night, whereas the colonies from Sept. 14 grew over two nights. Another plausible reason is that the centrifuge time was decreased to 5 min (instead of 8 min in the September 14 trials). The next step is to pray that these samples have the complete coding DNA sequence of PfDXR and have a 100% identity match. We will do another restriction enzyme test with HincII before sending (any) positive clones to DNA Sequencing.

//BLAST Sequencing Results//
 * September 19, 2012**

A2 pLIC-for 98% Max Ident. A2 pLIC-rev 99% Max Ident.

A4 pLIC-for 98% Max Ident. A4 pLIC-rev 99% Max Ident.

PfDXR Begins at base pair 155, ends at base pair 1621.

Insertions/Deletions:

A2-for, reliable from 134 to 982: 578 (deletion-C) 579 (deletion-G) 679 (insertion-C). //Insertion at 679 not seen in A2-rev.// A2-rev, reliable from 741 to 1673: 750 (deletion-T) 1525 (deletion-C), 1526 (deletion-G), 1528 (deletion-G), 1530 (deletion-A). //Deletion at 750 not seen in A2-for.//

A4-for, reliable from 78 to 982: 372 (insertion C) 558 (deletion-T) 560 (deletion-C) 561 (deletion-A) 562 (deletion-C) 720 (deletion-C). //Deletion at 720 IS seen in A4-rev.// A4-rev, reliable from 743 to 1683: No mutations. Will send another sample to DNA Sequencing A2-for: primer no. 9 A2-rev: primer no. 38

A4-for: primer no. 9 A4-rev: primer no. 38

Kaarthik - nice. - Dr. B 091812

//DNA Sequencing//
 * September 17, 2012**

Submitted 7 samples to the ICMB Core DSF (500 ng DNA each).


 * Week 2**

//Gel Analysis of Restriction Enzyme Digest (HincII) on pNIC-Bsa4 with Insert including controls// Lane 1: 1 kB DNA Ladder (5 ul) Lane 2: Control: pNIC-Bsa4 Uncut Lane 3: Control: pNIC-Bsa4 Cut with HincII Lane 4: alpha-1 pNIC-Bsa4 with Insert Cut with HincII Lane 5: alpha-2 pNIC-Bsa4 with Insert Cut with HincII Lane 6: alpha-3 pNIC-Bsa4 with Insert Cut with HincII Lane 7: alpha-4 pNIC-Bsa4 with Insert Cut with HincII Lane 8: beta-2 pNIC-Bsa4 with Insert Cut with HincII Lane 9: beta-3 pNIC-Bsa4 with Insert Cut with HincII Lane 10: tau-1 pNIC-Bsa4 with Insert Cut with HincII
 * September 14, 2012**

The Restriction Enzyme Digest positively confirms that the full Insert has been annealed to the accepting vector. Unfortunately, the gel was not run long enough (20 minutes, 130 V), resulting in a lackluster image. Fortunately, the digests on the Control (Lane 3) and the samples are distinguishable. There should be 3 cuts above 500 bp in the Control vector (Lane 3) according to the virtual digest available from NEBcutter. The top most band on Lane 3 may be the result of DNA sample from Lane 2 or DNA that was not cut. Meanwhile, it is also theorized that the pNIC-Bsa4 with insert will have 5 cuts, source: virtual digest from NEBcutter. What was expected correlates to the actual result, especially in alpha-2 and alpha-4 (Lane 4 and Lane 6, respectively). Overall, difference in band sizes indicates a successful transformation of pNIC-Bsa4. The next step is to verify the quality of the DNA amplification via DNA Sequencing.

pNIC-Bsa4 No Insert Virtual Digest (NEBcutter 2)

pNIC-Bsa4 DOXP Virtual Digest (NEBcutter 2)

//Nanodrop of Miniprepped pNIC-Bsa4 Insert//

Originally, we grew the //E. Coli// in 8 separate transformation tubes (alpha-1, alpha-2, alpha-3, alpha-4, beta-1, beta-2, beta-3, tau-1). Unfortunately, beta-1 had better things to do and died away, rest in peace. The alpha and beta colonies grew on two plates that contained sample B (48 ul Accepting Vector, 16 ul Insert) while the tau colony grew on a plate that contained sample A (2 ul Accepting Vector, 4 ul Insert). Only one colony grew from sample A (tau-1) while 15-20 colonies grew from sample B. The nanodrop results above indicate a very high concentration of pNIC-Bsa4 with Insert, showing promise for yield considerations. Additionally, the 260/230 and 260/280 absorbance values are in the recommended range, though higher 260/230 values would be more optimistic.

Another note: During the transformation step, 15 minutes passed by before SOC media was added after 30 minutes in ice. Additionally, the time in the shaking incubator was cut to 40 minutes.

//Accepting vector pNIC-Bsa4 digest verification with Gel Electrophoresis// Lane 1: Skip Lane 2: 1 kB DNA Ladder (5 ul) Lane 3: pNIC-Bsa4 cut with BsaI (AB) Lane 4: Skip Lane 5-6: pNIC-Bsa4 cut with BsaI (KR) Lane 7-10: Skip
 * September 11, 2012**

//PCR Clean Up pNIC-Bsa4// 260/280 ratio is 1.85, 260/230 ratio is 2.29. Concentration is 49.8 ng/ul. Sample is usable as an accepting vector.

260/280 ratio is 1.90, 260/230 ratio is 2.34. Concentration is 68.1 ng/ul. Sample is usable as an accepting vector.


 * Week 1**

//PCR^2 cleanup Nanodrop// Absorbance ratios are 1.89 at 260/280 (indicative of pure DNA) and 1.99 at 260/230 (another indication of pure DNA). Concentration is 105.0 ng/ul, suitable for inserting vector preparation and bacteria transformation.
 * September 07, 2012**

//Gel check of all PCR samples to determine viabilit////y.// Lane 1: Primary alpha - PCR done on 09.06.12 Lane 2: Primary gamma - PCR done on 09.06.12 Lane 3: Primary alpha - PCR done on 09.04.12 Lane 4: Primary beta - PCR done on 09.04.12 Lane 5: Primary gamma - PCR done on 09.04.12 Lane 6: Primary Sample A Lane 7: Primary Sample C Lane 8: Secondary gamma - PCR done on 09.06.12 Lane 9: Secondary from 08/01 sample Lane 10: Secondary from 07/18 sample

All samples seem to be okay, other than Lane 9 DNA sample.


 * September 06, 2012**

Gel check of secondary PCR^2 from September 04, 2012 - unsuccessful. Samples were left at 4 degrees Celsius. Tested Q5 High Fidelity Hot Start Polymerase with recommended NEB guidelines for primary PCR (20 cycles) - successful. Secondary PCR with Q5 HF was done (KOD thermocycling protocol) - successful. Lane 1: skip Lane 2: 1 kb DNA Ladder Lane 3-6: secondary PCR^2 (5 ul) KOD protocol Lane 7-8: skip Lane 9: ADO primary Lane 10: ADO secondary

Lane 1: Skip Lane 2: 1 kb DNA Ladder Lane 3: MM primary PCR Lane 4: MM secondary PCR Lane 5: KR primary PCR (alpha) Lane 6: KR primary PCR (beta) Lane 7: KR primary PCR (gamma) Lane 8: skip Lane 9: skip Lane 10: skip

Lane 1: Skip Lane 2: 1 kB DNA Ladder (5 ul) Lane 3: PCR^2 Sample ? Lane 4: PCR^2 Sample ? Lane 5-10: Skip


 * September 04, 2012**

Tested Q5 High Fidelity Hot Start Polymerase with KOD thermocycling protocol. Primary (30 cycles) and Secondary PCR were done. Lane 1: Skip Lane 2: 1 kB DNA Ladder (5 ul) Lane 3: Primary PCR alpha (06/18/12) (5 ul) Lane 4: Primary PCR beta (07/03/12) (5 ul) Lane 5: Primary PCR gamma (08/01/12) (5 ul) Lane 6-10: Skip

Lane 1: Skip Lane 2: 1 kB DNA Ladder (5 ul) Lane 3: Secondary PCR alpha (06/18/12) (5 ul) Lane 4: Secondary PCR beta (07/03/12) (5 ul) Lane 5: Secondary PCR gamma (08/01/12) (5 ul) Lane 6-10: Skip

Both overlap extension PCR worked, indicating that the KOD protocol (slightly modified with higher denaturation temp. at 98 Centigrade, and higher extension temp. at 72 Centigrade. Lane 3 in the Secondary PCR trial failed, most likely to negligence when some sample was lost before thermocycling.

Tested Q5 High Fidelity Polymerase (No hot start) with thermocycle protocol given by the info. booklet.
 * August 29, 2012**

Lane 1: Skip Lane 2: 1kb DNA Ladder Lane 3: Alpha primary PCR Lane 4: Alpha secondary PCR Lane 5: Beta primary PCR Lane 6: Beta secondary PCR Lane 7: Gamma primary PCR Lane 8: Gamma secondary PCR
 * August 02, 2012**

Gel results above indicate poor amplification of DNA. Other researchers experienced poor results also, indicating the KOD polymerase was not of usable quality anymore.

All 12 samples did not contain DXR recombinant gene. Only tail-end primers were sequenced, indicating contamination by smaller DNA fragments.
 * August 01, 2012**

Submitted 12 samples from **July 27, 2012** to DNA Sequencing
 * July 30, 2012**

Restriction Enzyme PvuII Digest of Miniprep Samples Lane 1: Skip Lane 2: 1kb DNA Ladder Lane 3: Control pNIC-Bsa4 No Insert Lane 4: pNIC-Bsa4 A3 No.1 Insert Lane 5: pNIC-Bsa4 A4 No.2 Insert Lane 6: pNIC-Bsa4 A4 No.3 Insert Lane 7: pNIC-Bsa4 B4 No.4 Insert Lane 8: pNIC-Bsa4 A3 No.5 Insert Lane 9: pNIC-Bsa4 B4 No.6 Insert Lane 10: Skip Star Activity is visible in Lanes 9&10. Lane 1: Skip Lane 2: 1kb DNA Ladder Lane 3: Control pNIC-Bsa4 No Insert Lane 4: pNIC-Bsa4 A4 No.7 Insert Lane 5: pNIC-Bsa4 A4 No.8 Insert Lane 6: pNIC-Bsa4 A3 No.9 Insert Lane 7: pNIC-Bsa4 B3 No.10 Insert Lane 8: pNIC-Bsa4 B3 No.11 Insert Lane 9: pNIC-Bsa4 A3 No.12 Insert Lane 10: Skip Star Activity is visible in Lanes 6 thru 9.
 * July 27, 2012**

Incubated for 3 hours at 37 degrees Celsius. No heat-block treatment past the water-block. This may explain the star activity in several of the lanes above.

Centrifuged and retrieved 12 pellets. We purified the contents of the lysate and measured the concentrations, below. A3 No. 1 50 ul Tris-HCL - Nanodrop pNIC-Bsa4, Concentration //86.7 ng/ul//, 260/280 //1.99// 260/230 //1.89// A4 No. 2 50 ul Tris-HCL - Nanodrop pNIC-Bsa4, Concentration 78.5 ng/ul 260/280 1.82 260/230 1.14 A4 No. 3 50 ul Tris-HCL - Nanodrop pNIC-Bsa4, Concentration 55.4 ng/ul 260/280 2.03 260/230 1.79 B4 No. 4 50 ul Tris-HCL - Nanodrop pNIC-Bsa4, Concentration 53.1 ng/ul 260/280 1.86 260/230 1.71 A3 No. 5 50 ul Tris-HCL - Nanodrop pNIC-Bsa4, Concentration 92.2 ng/ul 260/280 1.90 260/230 1.64 B3 No. 6 50 ul Tris-HCL - Nanodrop pNIC-Bsa4, Concentration 63.0 ng/ul 260/280 1.78 260/230 1.22 A4 No.7 50 ul Nanopure H20 - Nanodrop pNIC-Bsa4, Concentration 122.3 ng/ul, 260/280 1.77 260/230 1.02 A4 No.8 50 ul Nanopure H20 - Nanodrop pNIC-Bsa4, Concentration 76.2 ng/ul, 260/280 1.82, 260/230 1.16 A3 No. 9 50 ul Nanopure H20 - Nanodrop pNIC-Bsa4, Concentration 66.4 ng/ul 260/280 2.00 260/230 1.58 B3 No. 10, 50 ul Nanopure H20 - Nanodrop pNIC-Bsa4, Concentration 79.0 ng/ul, 260/280 1.85, 260/230 1.24 B3 No. 11, 50 ul Nanopure H20 - Nanodrop pNIC-Bsa4, Concentration 80.6 ng/ul 260/280 1.92, 260/230 1.48 A3 No. 12, 50 ul Nanopure H20 - Nanodrop pNIC-Bsa4, Concentration 60.4 ng/ul 260/280 2.02, 260/230 1.91
 * July 26, 2012**

Made master-plate and 12 tubes to incubate for 16 hours.
 * July 25, 2012**

Prepared 15*1ml aliquots 1 mg/ml Kanamycin Stock. Few colonies are visible on the four plates grown yesterday (**July 23, 2012**)
 * July 24, 2012**

Annealed and Transformed PCR product 3 (214) and PCR product 4 (170) with newly cut pNIC-Bsa4 Vector (?) and AH pNIC-Bsa4 Vector.
 * July 23, 2012**

Restriction Enzyme Digest, Verification of PCR Product and Accepting Vector, More pNIC-Bsa4 cut Lane 1: Skip Lane 2: 1kb Ladder Lane 3: PCR^2 AH Lane 4: 1A Miniprep Sample **July 18, 2012** Lane 5: 1B Miniprep Sample **July 18, 2012**
 * July 20, 2012**

The stream of DNA in Lane 4 and Lane 5 indicate that we have ended up with extraneous DNA.

To prepare for another trial run, we analyzed newly made inserting PCR product and accepting vector. Lane 1: Skip Lane 2: 1kb Ladder Lane 3: PCR Clean Up 3? Lane 4: PCR Clean up 4? Lane 5: 072012 pNIC-Bsa4 Cut Vector Lane 6: 1A Miniprep Sample **July 18, 2012** Lane 7: 1B Miniprep Sample **July 18, 2012**

pNIC-Bsa4 cut (started at 133 ng/ul) concentration, 27.1 ng/ul

Annealing and Transformation Take 2, PCR strikes back Lane 1: Skip Lane 2: 1kb DNA Ladder Lane 3: AB pNIC Cut Lane 4: AB pNIC Cut Lane 5: Skip Lane 6: KR Secondary PCR
 * July 19, 2012**

Nanodrop concentration: 213.9 ng/ul (eluted in 30 ul 10mM Tris HCl)

Nanodrop concentration: 170.2 ng/ul (eluted in 30 ul 10 mM Tris HCl)

We also submitted PCR Insert and pNIC-Bsa4 accepting vector (**July 16, 2012 and July 17, 2012)** after cohesive end generation to DNA sequencing to determine whether cohesive ends had been made before annealing and transformation.

PCR^2 Clean Up, Gel Analysis of Cut vector and PCR Insert (before and after cohesive ends). Nanodrop P.fal after PCR Clean Up 1A - Concentration 76.8 ng/ul, Absorbance ratios indicate pure DNA. //Contaminated// Nanodrop P.fal after PCR Clean Up 5B - Concentration 71.5 ng/ul, Absorbance ratios indicate pure DNA. //Contaminated//
 * July 18, 2012**

//July 27, 2012. The samples above did not contain the insert. The absorbance spectrums most likely indicate the presence of chromosomal DNA.//

Lane 1: Skip Lane 2: AB pNIC-Bsa4 Cut Lane 3: AB pNIC-Bsa4 Cut Lane 4: Skip Lane 5: PCR Clean Up 3? KR Lane 6: PCR Clean Up 4? KR Lane 7: pNIC-Bsa4 Cut (after cohesive end generation) Lane 8: PCR insert (after cohesive end generation)

Gel above revealed that PCR product and accepting vector were 'usable' - but does not demonstrate if cohesive ends had been formed.

Re Digest No. 2 with PvuII and Cohesive End Gel analysis Lane 1: 1 kb DNA Ladder Lane 2: AB pNIC Cut Lane 3: pNIC-Bsa4 (Uncut, no Insert) Lane 4: pNIC-Bsa4 (Cut with PvuII, no Insert) Lane 5-9: pNIC-Bsa4 (Cut with PvuII, with insert)
 * July 17, 2012**

We ran the restriction digest again to verify the findings from **July 16, 2012**. The results from DNA sequencing indicate no matches with the gene of interest.

RE Digest with PvuII and DNA Sequencing Lane 1: skip Lane 2: 1kb DNA Ladder Lane 3: pNIC-Bsa4 (no PvuII cut, no insert) Lane 4: pNIC-Bsa4 (after PvuII cut, no insert) Lane 5-9: pNIC-Bsa4 (after PvuII cut, with insert) Lane 10: skip
 * July 16, 2012**

Unexpectedly, there are no bands in Lanes 5-9. This indicates that vector with the gene insert had not been transformed, even though we witnessed colony growth on the master plate.

Samples were submitted for DNA sequencing.

Miniprep to extract and purify plasmid DNA from tubes 2 ul Sample 2 at 260 nm. //Contaminated// 2 ul Sample 2 Trial 2 at 260 nm. //Contaminated// 2 ul Sample 4 at 260 nm. //Contaminated// 2 ul Sample 4 Trial 2 at 260 nm. //Contaminated// 2 ul Sample 6 at 260 nm. //Contaminated// 2 ul Sample 6 Trial 2 at 260 nm. //Contaminated// 2 ul Sample 7 at 260 nm. //Contaminated// 2 ul Sample 7 Trial 2 at 260 nm. //Contaminated// 2 ul Sample 8 at 260 nm. //Contaminated// 2 ul Sample 8 Trial 2 at 260 nm. //Contaminated//
 * July 13, 2012**

Only colonies in tubes 2,4,6,7,8 produced a pellet after we centrifuged the conical tubes and removed supernatant. This is surprising since all tubes were incubated in similar conditions. We may have chosen 'bad' colonies (colonies were too large) in tubes 1,3,5. After plasmid preparation, the plasmid was eluted in 50ul Elution Buffer. The nanodrop results had concentrations between 25 ng plasmid/ul to 120 ng plasmid/ul. //July 27, 2012. None of the samples contained the insert. The high 260/230 absorbance ratios may indicate that samples have been contaminated. The samples in this trial were chosen from plates that contained many ( ~100 to 200 colonies), indicating that the kan. did not kill cells that did not transform.//

Protein Purification of FtHAP, Master Plate Growth of P.fal. Dxfr Elution 1 FtHAP Elution 2 FtHAP
 * July 12, 2012**

The nano drop measurements reveal very low absorbance values of protein. This is not surprising, as we deviated from protocol often.

8 colonies from the 2 plates transformed with the pNIC-Bsa4 vector were selected to grow in 8 tubes and one agar plate at 37 degrees Celsius.

Gel Check of pNIC-Bsa4 cut with BsaI after PCR clean-up Lane 1: Skip Lane 2: 1kb DNA Ladder Lane 3: 2.5 ul pNIC-Bsa4 digested with BsaI+ 0.5 ul blue dye Lane 4-10: Skip
 * July 11, 2012**

The two bands indicate two cuts were made, indicative of good result. Consequently, we made cohesive ends for the accepting vector (pNIC-Bsa4) and the inserting PCR DNA product (P.fal gene). The two pieces were annealed and transformed in 25 ul of DH5a Competent Cells and plated (2 plates) for overnight incubation.

Nanodrop pNIC-Bsa4 We cut the plasmid with the Bas1 Restriction Enzyme for 3 hours at 37 degrees Celsius. After PCR-Clean Up, we measured the concentration of plasmid - 40.8 ng/ul.
 * July 10, 2012**

Nanodrop P.fal. DXFR (Sample A) We did PCR Clean Up using the PCR^2 aliquots and measured the concentration of the DNA from scratch using the spectrophotometer. The average concentration (n=2) is 71.25 ng/ul.
 * July 09, 2012**

Secondary PCR and PCR^2 Lane 1: Skip Lane 1: Skip Lane 2: 1kb DNA Ladder Lane 2: 1kb DNA Ladder Lane 3: PCR^2 Aliquot 1 A Lane 3: PCR^2 Aliquot 1 C Lane 4: PCR^2 Aliquot 2 A Lane 4: PCR^2 Aliquot 2 C Lane 5: PCR^2 Aliquot 3 A Lane 5: PCR^2 Aliquot 3 C Lane 6: PCR^2 Aliquot 4 A Lane 6: PCR^2 Aliquot 4 C Lane 7: Skip Lane 7-10: Skip Lane 8: Skip Lane 9: Skip Lane 10: Skip
 * July 06, 2012**

Lane 1: Skip Lane 2: 1kb DNA Ladder Lane 3: Secondary PCR A Lane 4: Secondary PCR C Lane 5: Secondary PCR C (Sample tube was crushed in the thermocycler, also prematurely stopped final elongation). Lane 6: Skip Lane 7: Skip Lane 8: Skip Lane 9: Skip Lane 10: Skip

Both Secondary PCR and PCR^2 have worked.

Primary and Secondary PCR Lane 1: Skip Lane 2: 100 bp DNA Ladder Lane 3: 1kb DNA Ladder Lane 4: Primary PCR (PA) Lane 5: Secondary PCR Option B (PA) Lane 6: Skip Lane 7: Primary PCR, old Oligo Mix A (KR) Lane 8: Primary PCR, new Oligo Mix B (KR) Lane 9: Primary PCR, new Oligo Mix C (KR) Lane 10: Skip
 * July 05, 2012**

(Continuing from **July 02, 2012**) Comparing Lane 8 and Lane 9, Oligo Mix B was not properly prepared. Consequently, Oligo Mix B was discarded for future tests. The similarity in DNA product in Lane 7 and Lane 9 suggests that the old Oligo Mix was correctly prepared, but mistakes were made while preparing Secondary PCR.

Remade Oligo-Mix (two separate batches).
 * July 03, 2012**

Lane 1: Skip Lane 2: 1kb DNA Ladder, 5 ul Lane 3: Secondary PCR Option B, 15 ul Lane 4: Skip Lane 5: Skip Lane 6: Skip Lane 7: Skip Lane 8: Skip Lane 9: Skip Lane 10: Skip
 * July 02, 2012**

(Continuing from **June 29, 2012**)

The amplified Gene of Interest DNA is faintly visible in Lane 3. From the line matching up with the 1.5k bp, the band dims. Even though the band should be intense, it may be enough to validate the visible band from **June 29, 2012**.

Lane 1: Skip Lane 2: Skip Lane 3: 1kb DNA Ladder Lane 4: Primary PCR or Secondary PCR Option B? [KR] Lane 5: Primary PCR or Secondary PCR Option B? [KR] Lane 6: Skip Lane 7: Skip Lane 8: 100 bp DNA Ladder [UM] Lane 9: primary PCR [UM] Lane 10: secondary PCR B [UM]
 * June 29, 2012**
 * Looks good - hopefully the next round turns out better. - Dr. B**

(Continuing from **June 27, 2012**) In this attempt to clone the gene with custom primers, we changed the annealing temperature to 59 degrees celsius, diluted the custom primers in 10 ul final volume (previously made 5 ul final volume), and stored the PCR product in the -20 fridge immediately after the PCR cycling finished. However, the samples did not fluoresce well, evident above. Assuming the band in lane 3 is the 3kb DNA ladder fragment, then the band in lane 4 may be 1500 bp, which is the size of the gene we are studying.

We also monitored growth and harvesting of competent BL21 cells for protein expression.

Made 10x 50 ml and 5x 500 ml LB media for growing bacteria.
 * June 28, 2012**

Lane 1: 1 kb DNA Ladder Lane 2: primary PCR [AH] Lane 3: secondary PCR [AH] Lane 4: secondary PCR B new (but old custom primers) [KR] Lane 5: secondary PCR B new (but new custom primers) [KR] Lane 6: secondary PCR A new (but old Oligo Mix primers) [KR] Lane 7: secondary PCR A new (but new Oligo Mix primers) [KR} Lane 8: secondary PCR A old [KR] Lane 9: primary PCR new [KR] Lane 10: secondary PCR B old [KR]
 * June 27, 2012**

(Continuing from **June 26, 2012**) None of the PCR products fluoresced under UV. The faint stream in Lane 9 indicates that Primary PCR (with a newer sample of custom oligonucleotides) may have worked, but it is pale in comparison to Alex H. Primary PCR streak in Lane 2, indicating that it is not accurate.

Secondary PCR Overlap Option B Lane 1: Skip Lane 2: 1 kb DNA Ladder Lane 3: Skip Lane 4: Secondary PCR (Option B) KR Lane 5: Skip Lane 6: Skip Lane 7: Skip Lane 8: Skip Lane 9: Skip Lane 10: Skip
 * June 26, 2012**

(Continuing from **June 22, 2012)** Despite increasing the annealing temperature to 65 degrees Celsius, there is no resultant amplified DNA (would have been visible in Lane 4). This indicates that the product was not amplified. Either the template (primary PCR product) or the custom primers may be erroneous. (**June 27, 2012)** Annealing temperature is //not// 65 degrees Celsius; should be 60 degrees Celsius. See **June 22, 2012**.

PCR pNIC-Bsa4 Primers: pLIC-for and pLIC-rev
 * June 25, 2012**

Lane 1: Skip Lane 2: 100 bp DNA ladder 5 ul Lane 3: ? ng pNIC-Bsa4 KRAB Lane 4: ? ng pNIC-Bsa4 KRAB Lane 5: Control KRAB Lane 6: Skip Lane 7: ? ng pNIC-Bsa4 AH Lane 8: ? ng pNIC-Bsa4 AH Lane 9: ? ng pNIC-Bsa4 AH Lane 10: Control AH

Sample No. 3 KRAB (smallest amount of pNIC-Bsa4) is not shown above because it was misplaced after being taken out of the PCR machine.

Secondary PCR Overlap Option B Lane 1: Skip Lane 2: 1 kb DNA Ladder Lane 3: 100 bp DNA Ladder Lane 4: Primary PCR AB Lane 5: Secondary PCR (Option A) AB Lane 6: Secondary PCR (Option B) AB Lane 7: Secondary PCR (Option B) KR Lane 8: Skip Lane 9: Skip Lane 10: Skip
 * June 22, 2012**

Sample (prepared on **June 21, 2012**) was stored overnight at 4 degrees celsius. After electrophoresis the gel didn't want to get analyzed, so it slipped onto the ground. We then used 15 ul of the same sample on another gel, above. However, the DNA in this Lane 7 forgot to fluoresce. The annealing temperature may be suspect. We did not change the binding temperature from the Primary PCR. Since the melting temperature of the pNIC-Bsa4 custom primers is approximately 70 degrees Celsius, an annealing temperature of //65 degrees Celsius// may yield better results. (**June 27, 2012)** The previous statement is incorrect. We added 3 ul of 25 mM MgSO4 into 50 ul total volume, resulting in 1.5 mM MgSO4. The melting temperature of the custom primers is about 65 degrees Celsius at this concentration of Mg. The correct annealing temperature is around 60 degrees Celsius.

062112- Kaarthik. Nice job. Can you add a caption specifying the ladder size, your predicted gene size, and what the two different lanes are. Thanks, Dr. B

Primary PCR Overlap and Secondary PCR Overlap Option A Lane 1: Skip Lane 2: Skip Lane 3: 1 kb DNA Ladder Lane 4: Primary PCR AB Lane 5: Secondary PCR (Option A) AB Lane 6: Primary PCR KR Lane 7: Secondary PCR (Option A) KR (Approx. 1.5 kb). Lane 8: Skip Lane 9: Skip Lane 10: Skip
 * June 20, 2012**

The smear from the primary PCR is visible on Lane 6 and the secondary PCR product band is visible on Lane 7. It has a size of 1.5 kb. The actual gene size is 1467 bp.

PCR pNIC-Bsa4 Primers: pLIC-for and pLIC-rev Lane 1: 100 bp DNA ladder 5 ul Lane 2: 0.00824 ng pNIC-Bsa4 DB Lane 3: 0.0824 ng pNIC-Bsa4 DB Lane 4: 0.824 ng pNIC-Bsa4 DB Lane 5: 0.00824 ng pNIC-Bsa4 KR Lane 6: 0.0824 ng pNIC-Bsa4 KR Lane 7: 0.824 ng pNIC-Bsa4 KR Lane 8: Skip Lane 9: Skip Lane 10: Skip
 * June 20, 2012**

We used an annealing temperature of 48 degrees Celsius.

Taking a break.
 * June 19, 2012**

Made Oglio-Mix.
 * June 18, 2012**

Nanodrop pGBR22
 * June 15, 2012**

PCR pmCherry using VDS Primers and M13 Primers Lane 1: Skip Lane 2: 100 bp DNA ladder 5 ul Lane 3: 0.018 ng pmCherry VDS Set Lane 4: 0.18 ng pmCherry VDS Set Lane 5: 1.8 ng pmCherry VDS Set Lane 6: Skip Lane 7: 0.018 ng pmCherry M13 Set Lane 8: 0.18 ng pmCherry M13 Set Lane 9: 1.8 ng pmCherry M13 Set Lane 10: Skip
 * June 15, 2012**

There should be a dark distinct band instead of a faint band since Lane 9 contained 1.8 ng pmCherry. Maybe it jumped over to Lane 10.

Primer Design We designed a forward and reverse primer that will allow us to PCR the P.fal. gene and insert it into an expression vector (pNIC-Bsa4).
 * June 14, 2012**

__ Forward Primer: __ 5’ TACTTCCAATCCATGAAAAAGTATATCTACATCTAC 3’ 36 bp GC Content 30.6% 0 mM Mg2+ Tm 56.7 oC 1.5 mM Mg2+ Tm 64.9 oC 2 mM Mg2+ Tm 65.4 oC 4 mM Mg2+ Tm 66.5 oC 6 mM Mg2+ Tm 67.1 oC

__ Reverse Primer __ : 5’ AAACACAACTCTTCTTGA 3’ ** Reverse complement it: ** 5’ TATCCACCTTTACTGTCAAGAAGAGTTGTGTTT3’ 33 bp GC Content 36.4% 0 mM Mg2+ Tm 59.7 oC 1.5 mM Mg2+ Tm 67.6 oC 2 mM Mg2+ Tm 68.1 oC 4 mM Mg2+ Tm 69.2 oC 6 mM Mg2+ Tm 69.7 oC

PCR of pGBR22 using M13 Primers We repeated the experiment from **June 07, 2012** but used the M13 Primer Set instead of the SP6/T7 Primer Set. Lane 1: Skip Lane 2: 100bp DNA ladder Lane 3: 0.3 ng (1% conc.) pGBR22 plasmid KR Lane 4: 3 ng (1% conc.) pGBR22 plasmid KR Lane 5: 30 ng (10% conc.) pGBR22 plasmid KR Lane 6: Control. No pGBR22 plasmid Lane 7: 0.3 ng (1% conc.) pGBR22 plasmid AB Lane 8: 3 ng (1% conc.) pGBR22 plasmid AB Lane 9: 30 ng (10% conc.) pGBR22 plasmid AB Lane 10: Control. No pGBR22 plasmid. Band maybe contamination from Lane 9.
 * June 13, 2012**

Transformation of DH5alpha cells with pGFP NOTE: PLATE A AND C WERE REVERSED Plate A: 25ng of Plasmid, 224 colonies/ng
 * June 11, 2012**

Plate B: 5ng of Plasmid, 800 colonies/ng

Plate C: 1ng of Plasmid, 1200 colonies/ng

Amplify Purple Protein coding sequence in the pGBR22 plasmid using Forward and Reverse Primers PCR of pGBR22 using SP6/T7 Primers Lane 1: Skip Lane 2: 100bp DNA ladder Lanes 3-8: PCR Samples of pGBR22 using SP6/T7 Primers. No fragment bands are visible.
 * June 07, 2012**

We may have erred in pipetting or stoichiometric calculations. One plausible reason for the absence of any DNA fragments may be incorrect thermocycling programming (the annealing temperature entered corresponded to the M13 primer set, not the SP6/T7 primer set).

Restriction Digest Result Gel: Lane 1: Skipped Lane 2: DNA Ladder Lane 3: Uncut Plasmid AB Lane 4: EcoRI AB Lane 5: PvuII AB Lane 6: EcoR1+ PvuII AB Lane 7: EcoRI KR Lane 8: PvuII KR Lane 9: EcoR1+ PvuII KR Lane 10: Uncut Plasmid KR
 * June 07, 2012**