Grace+T.

Fall 2013

 * 12/04/13**

Enzyme Assay on Elution 1 of MabAFabG in M. tuberculosis in 15 mM HEPES buffer, 300 mM NaCl, and 5% glycerol. Spectrophotometer RedTide UV/Vis was used (Luke). Time-based readings at 60 reads/minute at 340.0 nm. This is the same for all 5 runs. Trial 1 Run 250 uL 15mM HEPES, 300 mM NaCl, and 5% glycerol; pH=7.57 150.5 uL Water 63.5 uL AAC [0.165 mM final] 26 uL NADPH [0.026 mM final] 10 uL Elution 1 MabA (+10 uL) x4 (+20 uL) x4 (+30 uL) x2 Total of 190 uL of enzyme

See minimal decline in NADPH absorbance, even though there was a lot of enzyme present. In the next run, should start with a higher initial enzyme concentration to see if a reaction occurs at all.

Trial 2 Run 250 uL 15mM HEPES, 300 mM NaCl, and 5% glycerol; pH=7.57 60.53 uL Water 63.5 uL AAC [0.165 mM final] 26 uL NADPH [0.026 mM final] 100 uL Elution 1 MabA

Steep decline seen witht he addition of enzyme, that is very steep, May be from active protein or just due to the change in total volume in cuvette.

(Blue) Trial 2 Run - Increased MabAFabG, Decreased water 250 uL 15mM HEPES, 300 mM NaCl, and 5% glycerol; pH=7.57 60.53 uL Water 63.5 uL AAC [0.165 mM final] 26 uL NADPH [0.026 mM final] 70 uL Elution 1 MabA

Another steep decline is seen, but there was a smaller decrease. It may be from active protein or just a volume change. In next run, dilute elution 1 to see if it effects the curve, making the line of decline less steep.



(Orange) Trial 4 Run - Dilute Elution 1 250 uL 15mM HEPES, 300 mM NaCl, and 5% glycerol; pH=7.57 90.5 uL Water 63.5 uL AAC [0.165 mM final] 26 uL NADPH [0.026 mM final] 70 uL Elution 1 MabA diluted to a 1:1

See a very broad line, slowly decreasing, but don't know why the start of decrease is higher than the initial NADPH reading. Redo Trial 4 to see if the same thing happens

(Purple) Trial 5 Run 250 uL 15mM HEPES, 300 mM NaCl, and 5% glycerol; pH=7.57 150.5 uL Water 63.5 uL AAC [0.165 mM final] 26 uL NADPH [0.026 mM final] 10 uL Elution 1 MabA A broad line of decrease higher than initial NADPH is seen. All absorbance values were higher in trial 5 than 4.


 * 11/27/13**

[[image:vdsstream/Gel_MabAFabG_112713.JPG width="423" height="320"]]
SDS Page-Protein gel ran at 200 V for 30 minutes, Lane 1: Protein DNA Ladder - BIOLADS New England Lane 2: Sample 1 - Cell Lysate After Induction Lane 3: Sample 2 - Soluble Faction Lane 4: Sample 3 - Flow Through Step Lane 5: Sample 4 - Wash Step Lane 6: Skip Lane 7: Sample 5 - Elution 1 of HEPES, 300 mM NaCl, and 5% glycerol Lane 8: Sample 6 - Elution 2 of HEPES, 300 mM NaCl, and 5% glycerol

Looks as if there is lots of contamination in Elution 1 ( or something wrong with gel) because there are bands all over the lane. May FPLC if enzyme assay shows strange results.

** -Week 13&14 **

 * 11/25/13**

Protein sample was sonicated and stored in Lysis Buffer. Syringe filtered protein and purified using nickel column, eluting in HEPES buffer of 300mm NaCl and 5% glycerol. Nanodropped to see concentration. ND1000 in A280 setting. Trial 1/2 of Elution 1 of MabAFabG in HEPES Buffer with 5% Glycerol. ND1000 in A280 setting. Trial 2/2 of Elution 1 of MabAFabG in HEPES Buffer with 5% Glycerol. ND1000 in A280 setting. Trial 1/2 of Elution 2 of MabAFabG in HEPES Buffer with 5% Glycerol. ND1000 in A280 setting. Trial 2/2 of Elution 2 of MabAFabG in HEPES Buffer with 5% Glycerol. Gel ran with Samples 1-6 and stained. (Will dry next week)

Midiprepped 2nd sample of protein in pNIC-Bsa4 plasmid. Stored in 3 different locations. ND1000 in Nucleic Acid setting. Trial 1/3 of midiprepped MabAFabG in pNIC-Bsa4. ND1000 in Nucleic Acid setting. Trial 2/3 of midiprepped MabAFabG in pNIC-Bsa4. ND1000 in Nucleic Acid setting. Trial 3/3 of midiprepped MabAFabG in pNIC-Bsa4.
 * 11/22/13**


 * 11/21/13**

Protein growth Midiprepped to make extra samples of cloned protein. ND1000 in Nucleic Acid setting. Trial 1/3 of midiprepped MabAFabG in pNIC-Bsa4. ND1000 in Nucleic Acid setting. Trial 3/3 of midiprepped MabAFabG in pNIC-Bsa4. Trial 2/3 was insignificant. Bad concentration obtained, so regrowth will be done. Second sample of MabAFabG in pNIC-Bsa4 was grown in LB + KAN. Large culture was made from small culture of MabAFabG in BL21(DE3) in LB Media and Kanamycin. Starting O.D. of 0.1 was grown in a 37 deg C shaking incubator until an O.D. of 0.5 is reached. Then grown at room temp. incubator for 18 hours. Sample was spun down and stored.

Small culture started with MabAFabG in BL21(DE3) cells. MabAFabG in pNIC-Bsa4 grown up in LB media with KAN.
 * 11/20/13**

Small culture started with untransformed protein ( MabAFabG in pNIC-Bsa4 instead of BL21(DE3) cells).
 * 11/13/13**

** -Week 11&12 **
Enzyme Assay: Time-based readings at 340.0 nm, ran with RedTide Spectrophotometer- UV rays; Optimal enzyme assay temperature = 25 deg C, with a pH of 7.6 Order of addition to cuvette: Buffer (50mM Tris), Water, AAC [0.165 mM], NADPH [0.026mM], and then enzyme (0.22mg/mL) Red: Trial 1: 250 uL Tris, 62.5 uL water, 82.5 uL AAC, 65 uL NADPH, and 40 uL of enzyme added at 3 minutes. + 40 uL of enzyme added at 5 minutes. Blue: Trial 2: 250 uL Tris, 62.5 uL water, 82.5 uL AAC, 65 uL NADPH, and 40 uL of enzyme added at 3 minutes. + 20 uL of enzyme added at 5 minutes, +20 at 6 minutes, +20uL of enzyme added at 6.8 minutes Green: Continuation from trial 2; +20 uL of enzyme added at beginning of run, +20 uL at 3 minutes, +20 uL at 3.7 minutes, +20 uL at 5 minutes, + 20uL at 6 minutes = totaling 200 uL of enzyme Orange: Trial 3 Change final NADPH concentration from 0.026mM to 0.04mM; 250 uL Tris, 27.5 uL water, 82.5 uL AAC, 100 uL NADPH, and 40uL of enzyme. Additional 240 uL of enzyme is added, for a total of 280 uL. Purple: Trial 4 Increase AAC concentration from 0.165 mM to 0.200 mM; 85 uL of enzyme added initially 250 uL Tris,100 uL AAC, 65 uL NADPH, and an initial 85 uL of enzyme. An additional 120 uL of enzyme was added to total 205 uL. Maroon: Trial 5 Increase AAC concentration from 0.165mM to 0.200mM; 250 uL Tris, 45 uL water, 100 uL AAC, 65 uL NADPH, and an initial amount of 40uL of enzyme. Another 690 uL of enzyme was added at different intervals, to total 730 uL.
 * 11/11/13 **

The enzyme didn't work. The decrease we see is most likely from an increase in volume, causing the absorption to decrease. Protein may be too dilute, so expression and purification will be redone with HEPES buffer instead of keeping the enzyme in Tris.


 * 11/7 **

Virtual Screening done on NIHCC Ligands (400) at autoscale 1 on 1 processor. Virtual Screening done on InHouseLigands (40) at autoscale 1 on 1 processor.
 * 11/6**

Virtual Screening done on CB306 (306) at autoscale 1 on 3 processors.
 * 11/4 Enzyme Assays **

Enzyme Assay: Time-based readings at 340.0 nm, ran with RedTide Spectrophotometer- UV rays; Optimal enzyme assay temperature = 25 deg C, with a pH of

7.6

Order of addition to cuvette: Buffer (50mM Tris), Water, AAC [0.165 mM], NADPH [0.026mM], and then enzyme (0.22mg/mL) Red: 1st Run; 250 uL Tris, 92.5 uL water, 82.5 uL AAC, 65 uL NADPH, and 10 uL enzyme added at 2.5 minutes. +10 ul enzyme at 3 minutes, +10 uL at 4 minutes, and +10 uL at 5 minutes Blue: 2nd Run; 250 uL Tris, 62.5 uL water, 82.5 uL AAC, 65 uL NADPH, and 40 uL of enzyme added at 3 minutes. Green: 3rd Run; wanted a straight line instead of exponential curve, made a 1:4 dilution of protein; 250 uL Tris, 62.5 uL water, 82.5 AAC, 65 uL NADPH, and 40 uL of diluted enzyme. Orange: 4th Run; ran ~60 minutes after initial run, used to check ability of NADPH and AAC to work after a long period of time; 250 uL Tris, 62.5 uL water, 82.5 AAC (old), 62 uL NADPH (old), and 40 uL of original enzyme Purple: 5th Run; ran with new NADPH (fresh), 250 uL buffer, 62.5 uL water, 82.5 uL AAC (old), 65 uL NADPH (fresh), and 40 uL original enzyme.

Analysis: Don't know what made run 2 to work when the first round didn't work so well. 3rd trial may have too dilute of enzyme; 4th and 5th trial indicates that fresh AAC and NADPH is necessary. Next step would be to try and reproduce trial 2, but to form a more linear line.


 * 11/1/13 **

Virtual screen ran with control ligands (22 ligands) on autoscale 2 on 1 processor. 3 ligands were unable to dock.

Scores were not as good as had hoped, lots of negatives ended up getting high scores while positives got average scores. However, the protein is able to go

through a virtual screening, so we will run it through different libraries.

** -Week 9&10 **

 * 11/7/13 - looks good. Try to get your assays repeatable. Dr. B **
 * 10/31 **
 * Spreadsheet for enzyme assay was produced, calculations of dilutions and concentrations within cuvette was done. Dilutions and aliquots were made for NADPH and AAC substrates. **


 * Next step: Start enzyme assay on 11/4 **


 * 10/30 **
 * Controls run was analyzed. Scores were obtained and categorized (belonging to positive, negative, or random control). **


 * Next step: Run protein through in-house library. **


 * 10/24 **
 * Controls were ligand prepped. Protein was prepped and the fake NADPH-substrate-substitute was removed. Active site was defined, and positive and negative controls were docked into said site. **


 * 10/23 **
 * NADPH was docked and used as a substrate to identify the active site. **

** -Week 7&8 **
Summary: Docking of NADPH of 1Q7B into 1UZN using Gold, Protein Expression (x3), Purification, and Characterization
 * Great work Grace. Nice job on the captions and analysis. Where exactly are you wit virtual work now? Thank you. -Max 10/21/13


 * 10/18**
 * Nanodrop of protein after FPLC was taken.**
 * ND1000 in A280 setting. Trial 1/2 of MabAFabG after FPLC, first peak shown stored at 4 degrees C.**
 * ND1000 in A280 setting. Trial 2/2 of MabAFabG after FPLC, first peak shown stored at 4 degrees C.**
 * ND1000 in A280 setting. Trial 1/2 of MabAFabG after FPLC, second peak shown stored at 4 degrees C.**
 * ND1000 in A280 setting. Trial 2/2 of MabAFabG after FPLC, second peak shown stored at 4 degrees C.**
 * ND1000 in A280 setting. Trial 1/2 of MabAFabG after FPLC, second peak shown stored at room temperature.**
 * ND1000 in A280 setting. Trial 2/2 of MabAFabG after FPLC, second peak shown stored at room temperature . **

ND1000 in A280 setting. Trial 1/2 of Elution 1 of MabAFabG in M. Tuberculosis in Elution Buffer. ND1000 in A280 setting. Trial 2/2 of Elution 1 of MabAFabG in M. Tuberculosis in Elution Buffer. ND1000 in A280 setting. Trial 1/2 of Elution 2 of MabAFabG in M. Tuberculosis in Elution Buffer. ND1000 in A280 setting. Trial 2/2 of Elution 2 of MabAFabG in M. Tuberculosis in Elution Buffer. Analysis: Really high concentration of protein after nickel column. May be overconcentrated perhaps. The elution 2 probably had more protein because elution 1 wasn't mixed up very well (Forgot to pipette up and down).
 * Analysis: Since there is really low concentration, must meet up with Dr. B to see if it is okay to go ahead with enzyme assay or if redoing the procedure in HEPES buffer is the better option. After 1 day of storage, the protein does not seem to be precipitating, will check up after weekend.**
 * 10/17**
 * Spun down and resuspended in Lysis Buffer, Sonication, Spin Down Step and Syringe Filtered. --Completion of Expression Protocol **
 * Nickel Column Purification, getting Elution 1 and 2**


 * Sample was concentrated down to 1mL for FPLC in Tris Buffer. However, after 30 minutes in the centrifuge, the protein started precipitating inside the concentrator. What was left of the protein was put into 1.7 mL centrifuge tubes and spun down to gather all protein that wasn't precipitating. The protein continued precipitation on the sides of tubes. Samples were syringe filtered and taken to FPLC. 1mL of protein was FPLC-ed, the rest were stored in 2 different conditions and nanodropped.**

ND1000 in A280 setting. Trial 1/2 of Syringe-Filtered sample of MabAFabG protein after purification and stored at room temperature. ND1000 in A280 setting. Trial 2/2 of Syringe-Filtered sample of MabAFabG protein after purification and stored at room temperature. ND1000 in A280 setting. Trial 1/2 of Syringe-Filtered sample of MabAFabG protein after purification and stored at 4 degrees C. ND1000 in A280 setting. Trial 2/2 of Syringe-Filtered sample MabAFabG protein after purification and stored at 4 degrees C.

FPLC of MabAFabG unconcentrated solution. 100 mL ran through column with Tris Buffer, collecting 2 peaks.


 * Analysis: 1mL of sample went through FPLC. Used the Nandini G75 Column, with Tris Buffer. Two peaks are seen, neither of which is at the 30 kDa mark, which is expected of the protein. After FPLC, the protein does not seem to be precipitating and is stored at both 4 degrees C and room temperature. Must reanalyze what storage buffer should be used so that the protein will not precipitate, maybe HEPES. Also, an ionic exchange may be done to further purify the protein. Could test enzyme assay now to figure out the situation with protein concentration and productivity.**


 * Protein was prepped for characterization.**

-Week 5&6**
 * 10/16**
 * Large Culture expression. O.D. reached, IPTG added and incubated flask in room temp. incubator for 20 hours.**
 * 10/15**
 * Small culture #3 for Protein Expression**
 * 10/11**
 * Docking of NAPH in 1Q7B, which has nice alignment with 1UZN. Next step for week 9: Use NADPH as a fake ligand to dock for the substrate in GOLD.**
 * 10/10**
 * Large Culture of Protein Expression; failed result because not enough protein was grown, and 20 mL in the tubes were not enough to bring protein O.D. up to 0.100 before placing in incubator.**
 * 10/9**
 * Redid Small Culture for Protein Expression**
 * 10/8**
 * Failed Expression #1, added too much KAN into cultures.**
 * Great captions and good analysis. Keep up the good work. Thank you. -Max 10/07/2013
 * Summary: Protein Expression, Purification, and Characterization**
 * Start of Virtual screening**

10/5
 * Positive controls and negative controls found**

10/4
 * Molecule search to find a structure that would allow us to insert a ligand close to the NADPH on our PDB struture of MabA (PDB: 1UZN). Unsuccessful, so we may have to place a ligand centered around an atom. **
 * FPLC sample concentrated and snap-frozen using liquid nitrogen.**
 * Gel characterization dried on vacuum.**
 * Protein PAGE gel dried**
 * Lane 1: Skipped to avoid smiling**
 * Lane 2: Loading Buffer (mistake. Should have used Protein Ladder) **
 * Lane 3: skip**
 * Lane 4: Sample 0 - Cell lysate before induction**
 * Lane 5: Sample 2 - Soluble fraction**
 * Lane 6: Sample 3 - flow through of sample in nickel column**
 * Lane 7: Sample 4 - wash through containing protein not bound to resin**
 * Lane 8: Sample 5 - Elution 1**
 * Lane 9: Sample 6 - Elution 2**
 * Lane 10: Skipped**


 * Protein PAGE gel of MabAFabG in M.Tuberculosis Expression**
 * Lane 1: Skipped to avoid smiling**
 * Lane 2: Loading Buffer (mistake. Should have used Protein Ladder) **
 * Lane 3: skip**
 * Lane 4: Sample 0 - Cell lysate before induction**
 * Lane 5: Sample 2 - Soluble fraction**
 * Lane 6: Sample 3 - flow through of sample in nickel column**
 * Lane 7: Sample 4 - wash through containing protein not bound to resin**
 * Lane 8: Sample 5 - Elution 1**
 * Lane 9: Sample 6 - Elution 2**
 * Lane 10: Skipped**

10/3
 * FPLC run complete, 3 different samples collected.**
 * FPLC run continued manually for 60 mL, making the full run of 130 mL. One peak is seen at the very end of the run, making our protein smaller than expected.**


 * Analysis: We see 3 bands. Either our protein is incomplete/mutated and very small, or we could have gotten very small yield. We could redo protein expression in different condition (growing it at room temperature instead of incubation at 37 degrees to see if there is better protein yield. It may also be possible that the large protein seen is caused from our protein clumping together or to the buffer itself. In the literature, purification was done in a phosphate buffer. If our buffer was changed or has more salt in it, there is a possibility that the protein will not clump and exist in its pure form.**

10/2
 * FPLC ran, but stopped too early, incomplete. 2 peaks shown.**


 * FPLC of MabAFabG, collecting 70 mL of concentration, showing two large peaks. Peaks came early in the run, so the protein is either very large or very small protein from the last FPLC run.**
 * Sample 2 was concentrated to 1 mL.**
 * Nanodrop of concentrated Elution 1 of Sample 2, Trial 1/2, of MabAFabG in M. Tuberculosis.**
 * Nanodrop of concentrated Elution 1 of Sample 2, Trial 2/2, of MabAFabG in M. Tuberculosis.**

9/30
 * Nickel column purification of Sample 2, stored for FPLC**
 * Nanodrop of Elution 1 of Sample 2, Trial 1/3, of MabAFabG in M. Tuberculosis.**
 * Nanodrop of Elution 1 of Sample 2, Trial 2/3, of MabAFabG in M. Tuberculosis.**
 * Nanodrop of Elution 1 of Sample 2, Trial 3/3, of MabAFabG in M. Tuberculosis.**
 * Nanodrop of Elution 2 of Sample 2, Trial 1/2, of MabAFabG in M. Tuberculosis.**
 * Nanodrop of Elution 2 of Sample 2, Trial 2/2, of MabAFabG in M. Tuberculosis.**

9/28
 * Nickel column purification of Sample 1, glycerol storage for long-term storage**
 * Nanodrop of Elution 1 of Sample 1, Trial 1/3, of MabAFabG in M. Tuberculosis.**
 * Nanodrop of Elution 1 of Sample 1, Trial 2/3, of MabAFabG in M. Tuberculosis.**
 * Nanodrop of Elution 1 of Sample 1, Trial 3/3, of MabAFabG in M. Tuberculosis. **
 * Nanodrop of Elution 2 of Sample 1, Trial 1/2, of MabAFabG in M. Tuberculosis.**
 * Nanodrop of Elution 2 of Sample 1, Trial 2/2, of MabAFabG in M. Tuberculosis.**

9/27
 * MabA Spin down step and Syringe Filter Step**
 * Move on to Nickel Column Purification**

9/26
 * Sonication of protein and stored in -20 degrees**

9/25
 * Large culture and Induction of protein expression with IPTG**
 * O.D. at .5 after 2.5 hours**
 * Concentrated protein stored in 2 15 mL conical tubes of Lysis Buffer.**

9/24 9/23 -Week 3&4 Grace - good work! Dr. B 100113** Summary: cloning completion and pNIC production 9/18 Cloning Results and BLAST DNA Sequencing results for all 8 samples were BLASTed against only the MabA (FabG) MTub Sequence. Sample 1 sequenced with the pLIC-Forward primer got a 99% identity and 33% query cover match. Sample 2 sequenced with the pLIC-Forward primer got a 93% identity and 20% query cover match. Sample 3 sequenced with the pLIC-Forward primer got a 99% identity and 33% query cover match. Sample 4 sequenced with the pLIC-Reverse primer got a 99% identity and 33% query cover match. Sample 5 sequenced with the pLIC-Reverse primer got a 98% identity and 79% query cover match. Sample 6 sequenced with the pLIC-Forward primer got a 100% identity and 13% query cover match. Sample 7 sequenced with the pLIC-Forward primer got a 99% identity and 13% query cover match. Sample 7 sequenced with the pLIC-Reverse primer got a 99% identity and 99% query cover match. Sample 8 sequenced with the pLIC-Forward primer got a 98% identity and 98% query cover match. Sample 8 sequenced with the pLIC-Reverse primer got a 99% identity and 99% query cover match.
 * One colony of transformed protein was innoculated in LB Media overnight for about 16 hours.**
 * Transformation of pNIC-Bsa4 into BL21(DE3) cells in order to make a competent cell optimal for protein expression.**
 * 2 plates were grown overnight.**
 * Plate of 10uL of MabAFabG of M.Tuberculosis in BL21(DE3) cells in LB Agar and KAN Plates.**
 * Plate of 50uL of MabAFabG of M.Tuberculosis in BL21(DE3) cells in LB Agar and KAN Plates.**

Analysis: Cloning was successful in clone #7 because all of the base pairs matched using either the pLIC-Reverse or the pLIC-Forward promers. Now we can move forward with protein expression.

9/13 RE Digest and Gel RE Digest gel of pNIC-Bsa4 and pNIC-Bsa4 with PCR insert Lane 1: 1 Kb DNA ladder from BIoLads NE Lane 3: Uncut pNIC-Bsa4 plasmid, pNIC control Lane 4-10: Sample 1-8 of MabA(FabG) in M.Tuberculosis in LB Media cut with PvuII and HINCII

DNA Sequencing Analysis: One clear band for all of the cloning sample is seen after RE digest. However, there are also 2 very light bands that can be seen (not so much on image, but under the UV light). Therefore, we can say that some, or even most of the clones had 3 bands, symbolizing that they were properly cut and MabA (FabG) was properly inserted into pNIC-Bsa4 in place of SacB. DNA sequencing is needed to have a definite confirmation of cloning success.

9/12 Observation of Masterplate& Miniprep Masterplate of MabAMTub in pNIC-Bsa4 on an LB Agar +KAN+ Sucrose Plate after 1 night incubation

Spin Down Nanodrop of Miniprep Samples: Nanodrop 1000 in Nucleic Acid setting. Trial 1/2 of Sample 1 of FabG (MabA) of M. Tuberculosis in pNIC-Bsa4. Nanodrop 1000 in Nucleic Acid setting. Trial 2/2 of Sample 1 of FabG (MabA) of M. Tuberculosis in pNIC-Bsa4. Nanodrop 1000 in Nucleic Acid setting. Trial 1/2 of Sample 2 of FabG (MabA) of M. Tuberculosis in pNIC-Bsa4. Nanodrop 1000 in Nucleic Acid setting. Trial 2/2 of Sample 2 of FabG (MabA) of M. Tuberculosis in pNIC-Bsa4. Nanodrop 1000 in Nucleic Acid setting. Trial 1/2 of Sample 3 of FabG (MabA) of M. Tuberculosis in pNIC-Bsa4. Nanodrop 1000 in Nucleic Acid setting. Trial 2/2 of Sample 3 of FabG (MabA) of M. Tuberculosis in pNIC-Bsa4. Nanodrop 1000 in Nucleic Acid setting. Trial 1/2 of Sample 4 of FabG (MabA) of M. Tuberculosis in pNIC-Bsa4. Nanodrop 1000 in Nucleic Acid setting. Trial 2/2 of Sample 4 of FabG (MabA) of M. Tuberculosis in pNIC-Bsa4. Nanodrop 1000 in Nucleic Acid setting. Trial 1/2 of Sample 5 of FabG (MabA) of M. Tuberculosis in pNIC-Bsa4. Nanodrop 1000 in Nucleic Acid setting. Trial 2/2 of Sample 5 of FabG (MabA) of M. Tuberculosis in pNIC-Bsa4. Nanodrop 1000 in Nucleic Acid setting. Trial 1/2 of Sample 6 of FabG (MabA) of M. Tuberculosis in pNIC-Bsa4. Nanodrop 1000 in Nucleic Acid setting. Trial 2/2 of Sample 6 of FabG (MabA) of M. Tuberculosis in pNIC-Bsa4. Nanodrop 1000 in Nucleic Acid setting. Trial 1/2 of Sample 7 of FabG (MabA) of M. Tuberculosis in pNIC-Bsa4. Nanodrop 1000 in Nucleic Acid setting. Trial 2/2 of Sample 7 of FabG (MabA) of M. Tuberculosis in pNIC-Bsa4. Nanodrop 1000 in Nucleic Acid setting. Trial 1/2 of Sample 8 of FabG (MabA) of M. Tuberculosis in pNIC-Bsa4. Nanodrop 1000 in Nucleic Acid setting. Trial 2/2 of Sample 8 of FabG (MabA) of M. Tuberculosis in pNIC-Bsa4. Analysis: Nanodrop shows a lower nucleotide concentration than round 3 of cloning, but this is not necessarily a negative factor. The highest values were from sample 7 and sample 8. Next step is RE Digest and DNA Sequencing. The masterplate showed high and concentrated growth which will be stored for later use.

9/11 Masterplates and Transformation tubes made

9/9 Observation of plate growth after Transformation: Plates of MabAMTub in pNIC-Bsa4 in a 1:2 ratio on LB Agar+KAN+Sucrose plates.

Plates of MabAMTub in pNIC-Bsa4 in a 1:1 ratio on LB Agar + KAN + Sucrose plates.

Analysis: Many colonies grew with the original protocol ratio of vector to insert. Although there were less clones than round 3 of cloning, this may be a good thing because the bacterial colonies are clear and distinct. The 1:1 ratio produced very little colonies, mainly to the side (so can't really be seen).

=**--Week 1&2**= Grace - ok your pNIC-Bsa4 might be good but cut longer next time. -- Dr. B 090913 Summary: Analysis of previous cloning, pNIC-Bsa4 transformation, Restarted cloning, secondary PCRs (making extras), and PyMol Refresher 9/8 Annealing and Transformation Cohesive End Generation for PCR Inserts and Accepting Vector PCR Clean-Up of pNIC-Bsa4 Nanodrop and gel Nanodrop 1000 in Nucleic Acid setting. Trial 1/2 of pNIC-Bsa4 accepting vector cut with BsaI-HF after PCR Clean-Up. Nanodrop 1000 in Nucleic Acid setting. Trial 2/2 of pNIC-Bsa4 accepting vector cut with BsaI-HF after PCR Clean-Up.

Agarose gel of cut pNIC-Bsa4 using RE BsaI-HF to remove 2000 bp SacB Lane 1: BioLad NE 100bp DNA Ladder Lane 2: pNIC-Bsa4 cut with BsaI-HF to remove SacB

Analysis: Concentrations of pNIC were consistent between the two trials and a lot higher than our first cloning attempt. The gel shows that BsaI-HF did cut properly because there are two visible bands. The lower band is the SacB portion, which is about 2000 bp. The higher band would be the pNIC-Bsa4 backbone without SacB. The bands on top could be contamination, or it could be pNIC-Bsa4 that has not been cut. To fix this problem, we have to leave it in the water bath for a longer amount of time in order to allow the BsaI-HF to complete its cutting.

9/6 Preparation of pNIC-Bsa4 as Accepting Vector for a 2nd time (because the first vector was mysteriously lost). Stored before PCR clean-up was done. 9/5 Preparation of pNIC-Bsa4 as Accepting Vector. Stored before PCR clean-up was done in -20 degrees freezer. Spin down pNIC-Bsa4 from Transformation and Midiprepped. Nanodrop: Nanodrop 1000 in Nucleic Acid setting. Trial 1/2 of DNA (pNIC-Bsa4) elution after Midiprep. Nanodrop 1000 in Nucleic Acid setting. Trial 2/2 of DNA (pNIC-Bsa4) elution after Midiprep. Analysis: Nanodrop concentration was extremely low. Even if I could verify that the product was pNIC-Bsa4, the concentration is almost useless, and not worth DNA sequencing. Possible errors include picking techniques, a bad colony selected, or too much time in the incubator. More pNIC-Bsa4 should be grown with less incubation time.

9/4 Grew pNIC-Bsa4 colonies from pre-made plates.

9/1 PyMol Refresher

8/30 Previous cloning results DNA Sequencing results for all 8 samples were BLASTed against the full pNIC-Bsa4 with MabAMTub Insert. Sample 1 sequenced with the pLIC-Forward primer got a 98% identity and 16% query cover match. Sample 2 sequenced with the pLIC-Forward primer got a 98% identity and 13% query cover match. Sample 3 sequenced with the pLIC-Forward primer got a 98% identity and 15% query cover match. Sample 4 sequenced with the pLIC-Reverse primer got a 98% identity and 15% query cover match. Sample 5 sequenced with the pLIC-Reverse primer got a 98% identity and 14% query cover match. Sample 6 sequenced with the pLIC-Forward primer got a 99% identity and 15% query cover match. Sample 7 sequenced with the pLIC-Reverse primer got a 99% identity and 13% query cover match. Sample 8 sequenced with the pLIC-Reverse primer got a 97% identity and 16% query cover match. Analysis: The identity matches were high, while the query cover was horrible. All of them have mutations, deletions, or random missing nucleotides. Also, the masterplate was lost over the summer, so cloning must be redone. RE Digest gel of pNIC-Bsa4 and pNIC-Bsa4 with PCR insert Lane 1: Sample 8: Tube 8 of pNIC and insert in LB Media cut with PvuII and HINCII Lane 2: 100 bp DNA ladder from BIoLads NE Lane 3: Uncut pNIC-Bsa4 plasmid, pNIC control Lane 4-10: Sample 1-7 of pNIC-Bsa4 and insert in LB Media cut with PvuII and HINCII

Analysis: We can see 4 bands in lanes 5-9, telling us that the PCR Inserts didn't make it into the pNIC-Bsa4 (refer to virtual gel for expected results). However, lane 1, 4, and 10 shows 3 bands. Waiting for DNA sequencing to see if any of them worked correctly and match our correct sequence. However, pNIC-Bsa4 control looks the most accurate and similar to the virtual gel. All the bands on the other lanes look off in terms of size placement.

Summer 2013 -Week 9
RE Digest using PvuII and HincII on pNIC-Bsa4 sample 1-8 with PCR Insert Virtual Gel: Virtual gel from NEB cutter. Gel of pNIC-Bsa4 cut with PvuII and HincII restriction enzymes. 4 bands expected on actual gel. Virtual gel from NEB cutter. Gel of pNIC-Bsa4 with PCR Insert cut with PvuII and HincII restriction enzymes. 3 bands expected on actual gel (Samples 1-8). Gel:

Result from transformation sent to DNA Sequencing DNA Sequencing Request form for pNIC-Bsa4 with FabG (MabA) PCR Insert. 8 samples were sent, 6 with pLIC-Forward and 2 with pLIC-Reverse primers.

--Week 8
Redid Cloning with 2 vector samples because the first samples had its temperature set to 65 degrees for about 5 minutes. We were worried it would kill the RE, so we ran another one just in case. Nanodrop and gel before clean-up Nanodrop 1000 in Nucleic Acid setting. Trial 1/2 of pNIC-Bsa4 vector #1 cut with BsaI-HF after PCR Clean-Up. Nanodrop 1000 in Nucleic Acid setting. Trial 2/2 of pNIC-Bsa4 vector #1 cut with BsaI-HF after PCR Clean-Up. Nanodrop 1000 in Nucleic Acid setting. Trial 1/2 of pNIC-Bsa4 vector #2 cut with BsaI-HF after PCR Clean-Up. Nanodrop 1000 in Nucleic Acid setting. Trial 2/2 of pNIC-Bsa4 vector #2 cut with BsaI-HF after PCR Clean-Up. Agarose Gel of pNIC-Bsa4 Accepting Vector cut with BsaI-HF Lane 1: PageRuler Protein Ladder Lane 2: Trial 2 of pNIC-Bsa4 vector #1 cut with BsaI-HF Lane 3: Trial 2 of pNIC-Bsa4 vector #2 cut with BsaI-HF The first vector had its bath temp. increase to 65 degrees for about 5 minutes, so we were scared that the enzyme had inactivated. Therefore, we have 2 samples.

2 vectors were made, so 2 separate cohesive ends generation were done with the vector and Inserts Positive bacterial growth, so we moved on to making a Master Plate and transformation tubes Spin down and Miniprep Nanodrop of Miniprep results of 8 samples Nanodrop 1000 in Nucleic Acid setting. Trial 1/2 of Sample 1 of pNIC-Bsa4 with FabG (MabA) PCR Insert. Nanodrop 1000 in Nucleic Acid setting. Trial 2/2 of Sample 1 of pNIC-Bsa4 with FabG (MabA) PCR Insert. Nanodrop 1000 in Nucleic Acid setting. Trial 1/2 of Sample 2 of pNIC-Bsa4 with FabG (MabA) PCR Insert. Nanodrop 1000 in Nucleic Acid setting. Trial 2/2 of Sample 2 of pNIC-Bsa4 with FabG (MabA) PCR Insert. Nanodrop 1000 in Nucleic Acid setting. Trial 1/2 of Sample 3 of pNIC-Bsa4 with FabG (MabA) PCR Insert. Nanodrop 1000 in Nucleic Acid setting. Trial 2/2 of Sample 3 of pNIC-Bsa4 with FabG (MabA) PCR Insert. Nanodrop 1000 in Nucleic Acid setting. Trial 1/2 of Sample 4 of pNIC-Bsa4 with FabG (MabA) PCR Insert. Nanodrop 1000 in Nucleic Acid setting. Trial 2/2 of Sample 4 of pNIC-Bsa4 with FabG (MabA) PCR Insert. Nanodrop 1000 in Nucleic Acid setting. Trial 1/2 of Sample 5 of pNIC-Bsa4 with FabG (MabA) PCR Insert. Nanodrop 1000 in Nucleic Acid setting. Trial 2/2 of Sample 5 of pNIC-Bsa4 with FabG (MabA) PCR Insert. Nanodrop 1000 in Nucleic Acid setting. Trial 1/2 of Sample 6 of pNIC-Bsa4 with FabG (MabA) PCR Insert. Nanodrop 1000 in Nucleic Acid setting. Trial 2/2 of Sample 6 of pNIC-Bsa4 with FabG (MabA) PCR Insert. Nanodrop 1000 in Nucleic Acid setting. Trial 1/2 of Sample 7 of pNIC-Bsa4 with FabG (MabA) PCR Insert. Nanodrop 1000 in Nucleic Acid setting. Trial 2/2 of Sample 7 of pNIC-Bsa4 with FabG (MabA) PCR Insert. Nanodrop 1000 in Nucleic Acid setting. Trial 1/2 of Sample 8 of pNIC-Bsa4 with FabG (MabA) PCR Insert. Nanodrop 1000 in Nucleic Acid setting. Trial 2/2 of Sample 8 of pNIC-Bsa4 with FabG (MabA) PCR Insert.

-Week 7
Begin Cloning Preparation of pNIC-Bsa4 done with triple the protocol ratio (plasmid amount stays the same) in 2 samples, later combined. PCR-Clean-up, Nanodropped, and a gel was ran. Nanodrop 1000 in Nucleic Acid setting. Trial 1/2 of pNIC-Bsa4 cut with BsaI-HF after PCR Clean-Up Nanodrop 1000 in Nucleic Acid setting. Trial 2/2 of pNIC-Bsa4 cut with BsaI-HF after PCR Clean-Up. Agarose Gel of pNIC Accepting Vector cut with BsaI-HF Lane 1: 100 bp DNA Ladder Lane 2: Trial 1 of pNIC-Bsa4 cut with BsaI-HF Second Preparation of pNIC-Bsa4 vector was done, this time with triple the ratio (except plasmid) and 3 samples, later combined. PCR-Clean up, and Nanodropped Nanodrop 1000 in Nucleic Acid setting. Trial 1/2 of pNIC-Bsa4 cut with BsaI-HF after PCR Clean-Up. Nanodrop 1000 in Nucleic Acid setting. Trial 2/2 of pNIC-Bsa4 cut with BsaI-HF after PCR Clean-Up

Cohesive End Generation was done on PCR Inserts and the pNIC-Bsa4 Accepting Vector Annealing and Transformation finished After overnight growth, samples placed in 4 degrees freezer because Master Plate can't be made unless a 2 day period is available.

Analysis: The first Nanodrop was too low, which signaled the need for the second preparation, this time with more samples, therefore more plasmid. Possible error: Protein was eluted with 50 uL of Elution Buffer from the Sigma Protocol instead of 30 uL called for in the cloning protocol. This could have led to a lower protein concentration when Nanodropping. The gel was only ran on the first pNIC-Bsa4 accepting vector because we don't want to waste vector on a gel. A protein Ladder should have been used instead of the 100bp DNA ladder. However, our protein did show two bands, meaning the BsaI-HF has cut the vector at 2 separate sites. On the next trial, a gel should be ran before doing PCR Clean-Up so that the final vector is not wasted.

-Week 6
RE Digest Gel: - This is the best RE digest so far! - Dr. B 071713 Probably re-do your pNIC-Bsa4 midiprep though Show your PCRs - overlap, secondary, etc.

Agarose gel of pGBR22 RE Digest on 7/8/13 Lane 1: 1 kb DNA Ladder from NE BioLads Lane 2: Uncut pGBR22 plasmid Lane 3: pGBR22 cut with EcoRI restriction enzyme Lane 4: pGBR22 cut with PvuII restriction enzyme Lane 5: pGBR22 cut with both EcoRI and PvuII There are clear and distinct bands in all 3 lanes with REs. EcoRI cuts in one specific site, whereas PvuII cuts at 2 locations. When we have both enzymes, we have 3 sites for enzyme cutting. Therefore, there are 1, 2, and then 3 bands. RE Digest worked well.

Primer Dilution made with MtubMabA Forward and Reverse Primer to make 100 uM from powder, ready for use or to make working stock. Overnight growth of pNIC-Bsa4 (bacteria colonies and transformation already pre-made) LB with bacteria spun down into a pellet and MidiPrep was done with pNIC-Bsa4. After Midiprep --> Nanodrop and DNA Sequencing Nanodrop 1000 in Nucleic Acid setting. Trial 1/4 of DNA (pNIC-Bsa4) elution after Midiprep. Nanodrop 1000 in Nucleic Acid setting. Trial 2/4 of DNA (pNIC-Bsa4) elution after Midiprep. Nanodrop 1000 in Nucleic Acid setting. Trial 3/4 of DNA (pNIC-Bsa4) elution after Midiprep. Nanodrop 1000 in Nucleic Acid setting. Trial 4/4 of DNA (pNIC-Bsa4) elution after Midiprep.

DNA Sequencing Request form for pNIC-Bsa4. 2 Samples sent, one with pLIC-Forward and one with pLIC-Reverse primer. (1 uL of primer and 11uL of plasmid) Growth of pNIC-Bsa4 in DH5alpha was done overnight, for 19 hours. Midiprep was troublesome because the MidiTip was clogged up and may not have been equilibrated properly. Different tip was used, but plasmid may have been lost in the process. Nanodrop values were very diverse and not precision. Accuracy is unclear, so a maximum amount of DNA was sent to sequencing.

2 Unverified plasmid from previous weeks were Nanodropped and sent to DNA Sequencing. Nanodrop 1000 in Nucleic Acid setting. Trial 1/2 of pmcherry elution (blue tab) from unverified plasmids (after Midiprep) Nanodrop 1000 in Nucleic Acid setting. Trial 2/2 of pmcherry elution (blue tab) from unverified plasmids (after Midiprep) Nanodrop 1000 in Nucleic Acid setting. Trial 1/2 of pmcherry elution (red tab) from unverified plasmids (after Midiprep) Nanodrop 1000 in Nucleic Acid setting. Trial 2/2 of pmcherry elution (red tab) from unverified plasmids (after Midiprep) DNA Sequencing Request Form for pmcherry. 2 samples were sent using M13-For-20 in-house primers Blue: 2.1uL plasmid + 8.9uL dH2O Red: 2.4uL plasmid + 8.6uL dH2O

Redo: Overnight Growth of pNIC-Bsa4 Because of the many errors, and unclear DNA sequencing results, pNIC-Bsa4 was redone. Overnight Growth of bacteria already pre-made. LB and plasmid spun down into pellets and then was MidiPrepped Plasmid was then Nanodropped and sent to DNA sequencing

Nanodrop 1000 in Nucleic Acid setting. Trial 1/2 of DNA elution (pNIC-Bsa4) after Midiprep Nanodrop 1000 in Nucleic Acid setting. Trial 2/2 of DNA elution (pNIC-Bsa4) after Midiprep DNA Sequencing Request form for pNIC-Bsa4. 2 Samples sent, one with pLIC-Forward and one with pLIC-Reverse primer. (1uL primer + 8.11uL pNIC + 2.89ul dH2O)

Oligo Mix for MtubMabA was made Primer Overlap with Q5 Polymerase Primary and Secondary PCR was done and gel was ran Gel: Agarose Gel of Overlap PCR of MtubMabA primers Lane 1: 1Kb DNA Ladder from NE BioLads Lane 2: Primary PCR Lane 3: Secondary PCR Nothing showed up, so our PCR's were unsuccessful. Redo of Primary and Secondary PCR Adjustment: change the anneal temperature, lowering it by 5 degrees C (53 degrees C) Gel ran: Agarose gel of Overlap PCR for MtubMabA primers Lane 1: 100 bp DNA Ladder from NE BioLads Lane 2: Primary PCR with Oligo Mix Lane 3: Secondary PCR with primary PCR and primers This second attempt was successful because there is a smear in lane 2 and a clear bright band in lane 3. We can estimate that the Gene Sequence has about 800 bp. We now that from our Primer design, the product should be 744 bp, so our gel is accurate. PCR squared, PCR clean up, and then Nanodropped: Nanodrop 1000 in Nucleic Acid setting. Trial 1/2 of PCR product after PCR clean-up.

Nanodrop 1000 in Nucleic Acid setting. Trial 2/2 of PCR product after PCR clean-up.

Since the Nanodropped concentration was too low to be appropriate if PCR was successful, PCR squared was redone. Adjustment: The anneal temperature was raised 5 degrees Celsius, from 53 to 58 degrees, which was the starting protocol. Nanodrop 1000 in Nucleic Acid setting. Trial 1/2 of PCR product after PCR clean-up.

Nanodrop 1000 in Nucleic Acid setting. Trial 2/2 of PCR product after PCR clean-up.

Purification of YopH protein was done. With the Elution 1 from protein purification of the previous week, FPLC was performed. All Elution 1 (4 groups) were spun down and concentrated, then stored in -20 degrees freezer. Because the protein was not stable, freezing it caused FPLC to be impossible. Therefore, FPLC was performed with ECDHFR and ran for about 2 hours. FPLC Results after about 1.5 hours running with ECDHFR. The product was combined and spun down again.

-Week 5
Gel from Pmcherry PCR Trial 5: Lane 1: 100 bp DNA Ladder Lane 2-9: Sample 1-8 No amplification, but vague band was seen in Lane 4 which contained 3, with 2.4 ng of pmcherry plassmid using VDSR1 and VDSR2 primers. Pattern is that only one lane shows up. Possible that there is not enough plasmid, that the pmcherry will only work with the VDSR1 and VDSR2 primers, or another technical PCR error. PCR of pGFP plasmid (green) Trial 1: Temp set at 5 degrees below Tm of Primers. Gel: Fig. 1: PCR 1% agarose gel; 7/2/13 Lane 1: 100 bp DNA Ladder from NE BioLabs Lane 2: Sample 1: 0.1 ng of pGFP DNA using VDS1 & VDS2 Primers Lane 3: Sample 2: 0.6 ng of pGFP DNA using VDS1 & VDS2 Primers Lane 4: Sample 3: 5.7 ng of pGFP DNA using VDS1 & VDS2 Primers Lane 5: Sample 4: No DNA; Control Lane Lane 6: Sample 5: 0.1 ng of pGFP DNA using M13 Forward & M13 Reverse Primers Lane 7: Sample 6: 0.6 ng of pGFP DNA using M13 Forward & M13 Reverse Primers Lane 8: Sample 7: 5.7 ng of pGFP DNA using M13 Forward & M13 Reverse Primers Lane 9: Sample 8: No DNA; Control Lane Contamination seen in Lane 9, which should not have had any bands. Different primers cut the DNA at different parts, so the sizes of the DNA are different depending on the primers. The VDS1 and VDS2 primers gave us DNA with about 1,500 bps. The M13 primers gave us DNA with about 1,000 bp. Protein Sonication (2nd time) and Spin Down Step Protein Purification Nanodrop of Elutions: ND1000 program in A280nm setting for protein reading. Sample was from Elution 1, containing Elution buffer and YopH protein.

ND1000 program in A280nm setting for protein reading. Sample was from Elution 2, containing Elution buffer and any leftover YopH protein that was not purified in Elution 1.

Nanodrop showed that our purification step worked, and all/most of the YopH protein is present in Elution 1, and barely any in Elution 2. Protein Characterization Step Homemade SDS Gel Ran and stained SDS Gel of protein, dried on 7/8/13 Lane 1: BioRad Protein Ladder Lane 2: Sample 0 - Cell lysate before induction Lane 3: Sample 1 - Cell lysate after induction Lane 4: Sample 2 - Soluble fraction Lane 5: Sample 3 - Flow through Lane 6: Sample 4 - Wash Lane 7: Sample 5 - Elution 1 Lane 8: Sample 6 - Elution 2 Lanes 2-6 has smears all over the place, representing the proteins that we have filtered out. Lane 7 and 8 should have only our YopH protein, but we can see a bit of contamination of a larger protein. Our protein is around 35 kDa.

Restriction Enzyme Digest was done on pGBR22. Will run gel next week.

Week 4
PCR of pmcherry plasmid (red) Trial 4: Temp set at 10 degrees Celsius below Melting Temp. of primers Gel:

[[image:vdsstream/gtpmcherry4.jpg width="320" height="370"]]
Lane 1: 100 bp DNA ladder Lane 2-9: Samples 1-8 No amplification, but vague band was seen in Lane 4 which contained 3, with 2.4 ng of pmcherry plassmid using VDSR1 and VDSR2 primers. PCR of pmcherry plasmid (red) Trial 5: Anneal temp set at 5 degrees below Tm of primers: Will run Gel next week. Protein Expression Scaled Up: Sonication and then Spin Down Step

Week 3
PCR of pmcherry plasmid (red) Trial 1: Temp set at 5 degrees Celsius below Tm Gel 1: NO AMPLIFICATION Error in Trial 1: Temperature of anneal step is probably too high PCR of pmcherry plasmid (red) Trial 2: Temp set 10 degrees Celsius below Tm Gel 2: NO AMPLIFICATION Error in trial 2: Ran for 35 cycles instead of 30

Protein expression stored in -80 degrees fridge. End weight: 1.01 g BL21(DE3) cells with YopH plasmid: LB Agar and KAN antibiotics with 10 uL of YopH bacteria grown in BL21(DE3) cells after overnight incubation (6/18/13) LB Agar and KAN antibiotics with 50 uL of YopH bacteria grown in BL21(DE3) cells after overnight incubation (6/18/13)

PCR pmcherry plasmid (red) Trial 3: Temp of anneal set at 10 degrees Celsius below Tm Gel 3: Error; Contamination, but amplification was shown! YAY PCR run on pLic protein Gel: Agarose 1% gel of pLIC-Bs4; 6/19/13 Lane 1: 100 bp DNA Ladder from New England BioLabs Lane 2: Sample 1: 0.035 ng of pLIC DNA Lane 3: Sample 2: 0.35 ng of pLIC DNA Lane 4: Sample 3: 3.5 ng of pLIC DNA Lane 5: Sample 4: DNA control with no plasmid It is seen that there are increasing brightness of bands as the amounts of pLIC DNA increase. The gel shows no contamination because the lanes have single bands with similar locations. Based on the ladder, we can predict that our pLIC DNA is about 1,200 bp. 100 bp DNA Ladder visualized by ethidium bromide staining on a 1.3% TAE agarose gel. Mass values are for 0.5 µg/lane.

-Week 2 - Grace - good work! - Dr. B
Overnight plasmid of pmcherry grown and spin down in DH5alpha cells PCR of pGBR22 plasmid (purple) [My 1st PCR] MidiPrep of spun down pmcherry.

Nanodrop of MidiPrep DNA final elution of pGBR22 plasmid, using ND1000 Nucleic Acid setting. Trial 1 out of 2.

Nanodrop of Midiprep DNA final elution of pGBR22 plasmid, using ND1000 Nucleic Acid setting. Trial 2 out of 2.

Order form for DNA sequencing of Midiprep final DNA elution of pGBR22 plasmid. Order number 91954.

1% Agarose gel of PCR: Agarose gel of pGBR22 plasmid after PCR, gel at 110 V. Lane 1: 100 bp DNA Ladder from New England Biolads Lane 2: Sample A, 0.3 ng of pGBR22 plasmid Lane 3: Sample B, 3 ng of PGBR22 plasmid Lane 4: Sample C, 30 ng of PGBR22 plasmid Lane 5: Sample D, Control, No DNA 100 bp DNA Ladder visualized by ethidium bromide staining on a 1.3% TAE agarose gel. Mass values are for 0.5 µg/lane.

PCR of red pmcherry plasmid ( now in freezer, will run gel next week)

--Week 1 Dilution of primer: M13 Reverse (-27) from 100 microM to 20 microM Nanodrop of green forward primer (pGFP): average = 4650.15 ng/microL DNA sequencing of pGFP requested; Order number 91628 4 plates of LB Agar Plates was made with Ampicillin on Agar Transformation Efficiency: pmcherry with a 120ng/microL Colonies grown overnight; pictures below First PCR with pGBR22; M13 Forward (-20) and M13 Reverse (-27) Primers; Stored in freezer box. Results: Plate A with 1 ng of pmCherry plasmid on LB Agar with Ampicillin. Picture taken on 6/6/13, after one night of incubating. 3150 colonies total, transformation efficiency of 3150 colonies/ng. Plate B with 5 ng of pmCherry plasmid on LB Agar with Ampicillin. Picture taken on 6/6/13, after one night of incubating. 3375 colonies total, transformation efficiency of 675 colonies/ng. Plate C with 25 ng of pmCherry plasmid on LB Agar with Ampicillin. Picture taken on 6/6/13, after one night of incubating.3510 colonies total, transformation efficiency of 140 colonies/ng.