Paul+P.

week 15:

12.6.12 Worked with ling to assay suman's protein.

The measured absorbance for 410nm was 0.  Since other people had similar results it could be reasoned that the cause for this would be: 1.multiple times of defrosting 2.the glycerol mix was bad 3. it was not stored in glycerol at all

Afterwards we measured Suman's protein STSP 2 more times. the first time it did not work because the values for the uM and molecular weight were incorrect, so the addition of tris base was incorrect. The second time, the correct values were entered, and that is when the above conclusions were made.

12-5-12 Enzyme assay with FtHap; Negative results as there is no absorbency measured at 410 nm which is the wavelength where the protein should present itself if there was indeed protein activity when introduced with the pNPP. reasons could be :

1. improper technique with pipetting and use of the wrong cuvette 2. the protein was inactive due to denaturing 3. not enough of the protein within the solution. The values (absorbance) were too small to consider using for inhibition. During the collection of absorbance, the samples got mixed up. Also there isn't a linear function as the concentrations of enzyme increased. Also, one of the samples was thrown out because it was skewed from the rest of the data.

12-4-12

Conducted enzyme assays with ling and rishi on the surrogate FtHap. Still getting inconclusive results with the redtide machine. More assays will be run in order to verify whether or not the issue is technique or the protein.

Conducted virtual screening 2nd run - results:



The results shown here are the top 10 ligands selected by the GOLD program out of 400 compounds from the HF9PlatesPlates library. Compounds HTS09290, SEW02685, and HTS08822 are the top 3 ligands from the selection of 400 and would make the best potential drug candidates out of the compounds screened. The compound 1 shows much promise with a fitness score of 99.23%.

Week 14: 11.30.12

Started work on virtual screening.

11.29.12 Worked on assaying / measuring concentration samples

Worked on swiss model:

11.27.12

Worked on purification protocols - used GE purification tubes in combination with the beckman centrifuge in order to remove water and extract a pure sample. Sample found to be roughly around 0.98 mg/ml.

week 13:

112612 - ?? . Dr B

Characterized the surrogate with rishi - <span style="background-color: #ffffff; color: #000000; font-family: arial,helvetica,sans-serif; font-size: 13px; text-align: start;">Nanodrop/concentration pulled from rishi's vds page. <span style="background-color: #ffffff; color: #000000; font-family: arial,helvetica,sans-serif; font-size: 13px; text-align: start;">Lane 1: Skipped <span style="background-color: #ffffff; color: #000000; font-family: arial,helvetica,sans-serif; font-size: 13px; text-align: start;">Lane 2: 10-170kDa Protein Ladder <span style="background-color: #ffffff; color: #000000; font-family: arial,helvetica,sans-serif; font-size: 13px; text-align: start;">Lane 3: Sample 1, Cell lysate after induction <span style="background-color: #ffffff; color: #000000; font-family: arial,helvetica,sans-serif; font-size: 13px; text-align: start;">Lane 4: Sample 2, Soluble fraction <span style="background-color: #ffffff; color: #000000; font-family: arial,helvetica,sans-serif; font-size: 13px; text-align: start;">Lane 5: Sample 3, Flow through <span style="background-color: #ffffff; color: #000000; font-family: arial,helvetica,sans-serif; font-size: 13px; text-align: start;">Lane 6: Sample 4, Wash <span style="background-color: #ffffff; color: #000000; font-family: arial,helvetica,sans-serif; font-size: 13px; text-align: start;">Lane 7: Elution 1 from Trial 1 (Tube A) <span style="background-color: #ffffff; color: #000000; font-family: arial,helvetica,sans-serif; font-size: 13px; text-align: start;">Lane 8: Elution 2 from Trial 1 (Tube B) <span style="background-color: #ffffff; color: #000000; font-family: arial,helvetica,sans-serif; font-size: 13px; text-align: start;">Lane 9-10: Skipped
 * <span style="background-color: #ffffff; color: #000000; font-family: arial,helvetica,sans-serif; font-size: 13px; text-align: start;">Some of Elution 2 spilled into Lane 9 causing faded bands. Also, Cell Lysate before induction was not inserted into Lane because not available. **

<span style="background-color: #ffffff; color: #000000; font-family: arial,helvetica,sans-serif; font-size: 13px; text-align: start;">Elution 1 (Concentration of 1.40 mg/mL): Tube A from Trial 1
 * [[image:vdsstream/Elution 1ubeB_RDvds.jpg caption="Elution 1ubeB_RDvds.jpg"]] ||
 * Elution 1ubeB_RDvds.jpg ||

<span style="background-color: #ffffff; color: #000000; font-family: arial,helvetica,sans-serif; font-size: 13px; text-align: start;">Elution 2 (Concentration of 0.11 mg/mL): Tube B from Trial 1
 * [[image:vdsstream/Elution 1tube A_RDvds.jpg caption="Elution 1tube A_RDvds.jpg"]] ||
 * Elution 1tube A_RDvds.jpg ||

<span style="background-color: #ffffff; color: #000000; font-family: arial,helvetica,sans-serif; font-size: 13px; text-align: start;">Elution 1 (Concentration of 1.08 mg/mL): Tube C from Trial 2
 * [[image:vdsstream/Elution 2tubeC_RDvds.jpg caption="Elution 2tubeC_RDvds.jpg"]] ||
 * Elution 2tubeC_RDvds.jpg ||

<span style="background-color: #ffffff; color: #000000; font-family: arial,helvetica,sans-serif; font-size: 13px; text-align: start;">Elution 2 (Concentration of 0.10 mg/mL): Tube D from Trial 2
 * [[image:vdsstream/Elution 2tubeD_RDvds.jpg caption="Elution 2tubeD_RDvds.jpg"]] ||
 * Elution 2tubeD_RDvds.jpg ||

Week 12: 11.18.12 Assisted Rishi on Sonification steps.

Out put from the sequencing of lmstpp target: " code NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGNNCTGANNNNNNNNNNNNNNANNNNGANGNANNAAAANNANNNNNNN NGNNNNNTNNNNNNNNNTCTCNTNNNGACTCGNNNNNNNNNNNANTGACANCATNGATCCTGNNNCATATGCATAANGGN NNNGTAAGGNTCCNTACNNGACTGCNCNAAANCAACCNNACNNTGCNTGTNTCCNCTCTGGNTTANTCATCGNCNTCNAT CTCTTAAAAAAGGGCTTCTTATCCTTTGGAAGTNANNCTGAAAAATTTGTNGCNATGCTTGAANTTGCGAAAANGGAAGA CCAANTTTNANTNGAAAGNCTTGAACTATACTTCACGTCTTTGTTGAAATNNGANAATTGACCCTCTCCCATACTTANNC TNNCNTTATCANTTGATTCTTCATTTTCCNTGNNTTGTAAGNNGANNNNNNTCNNGTNNNGANNATNTACATNCTCCATG AGCTCTNAGGTGATCCAGTTNCTACNATCGGACNATTTTNGAATGCNCGGNTGGCATCGACCTTACGAATANGCAANAGG CGGGAGNANTAACAATANNTGTACGANNNNANNGGNNNTGNNTAANGNNTCNNGGACTACTCTTTCNGANTNNNAATCTA ANGNNCTTGTNGCCTCGNCTNNNNNNANANNTGNNNNNNGATTNNANNANNNNNCGNGCNANNGAANTCNTTTNNNTTCT NACCCCCTGAAATTNNNNCCTCNNTCTCTCCCNNAACCNTNNTNTACACAANNATAANAACTANNTNNNNNNGTCTGANN NNGNTNANCANNNNNCNANTTTCNAAANGNTGNNNTCTANCCNGANNTTTNGGACCCAGNANGGAANTGAANNGTGNNAN" code When entered onto blast, there was not enough of a match up to give conclusive results.

Week11: Paul - make sure you use the right primers for you sequencing. Not sure why you are getting unreadables. Maybe show me you output form one of them - post it here on Wiki. -- Dr. B 11/19/12

11.15.12 Morning to mid day was spent with the continued growth of the Fthap colonies in 2L flask ; 4 out of the 6 flasked were used due to space constraints. Every hr I measured OD for the flasked until 0.5 absorbency at 600 nm was reached. Afterwards I added ITPG and left the flask in the shaker for 4 hours to allow it to grow. Subsequent centrifugal was done by ling and rishi after the 4 hour period.

11.14.12 I stayed over night with ling to work on growing up the fthap colonies for surrogate protein expression. 6 flasks in total were left in the shaker over night.

11.12.12 I looked over the data recieved from the core and checked against blast was inconclusive. There was a large number of N and unreadable nucleotides in the sequence. I will grow more bacteria this week and resend samples for sequecing to verify my results. As well I will now start on the surrogate protein expression.

Week10: 11.9.12

I am currently awaiting results of DNA sequencing.

11.8.12 Nanodrop concentration results : Samples 4 and 9 show really weak absorption/concentration. The reasons for this could be that those colonies did not grow too well in the media, improper growing technique, or improper minipreping. Next step would be to prepare the samples for sequencing.















11.7.12

Worked on miniprep of the colonies and extraction of dna. 11.6.12 I prepared 9 colonies for master plate production and grew up 9 tubes.

My everything plate has produced colonies - there are roughly around 60 colonies. My next step is to move onto picking a couple of colonies and proceed to making a master plate and producing some bacteria for sequencing.

Week9:

11.2.12

I have decided to employ a hail mary approach with my own samples while working with ling and rishi to help them get their samples to cloning. What I have done is mixed together 3-4 months worth of my own personal leftover and unused lmstpp and bsa-4 samples that were properly labeled into one tube of accepting plasmid and one tube of the insert. This produced a high concentration of plasmid to insert. I also ran a gel check to see if the samples were still use able. On a 100b ladder, both the insert and the accepting vector seem to be within the right size, length, and cut amounts. When plating I made a 20:20 ratio of accepting to insert - and added 50 ul of the bacteria to the mixture while prepping. As well checking the nanodrop readings, while the absorbances are high, the wavelengths they each respectively peak at are correct when compared to past samples.





11.1.12 Worked on PCR^2 and clean up with ling.



10.31.12

Made primer dilutions for the master stock and for working solutions. Attempted to do a midiprep clean up - failed horribly due to incorrect tubes being used.

10.29.12 Prepared samples for additional people for leishmania target.

week8:

10.25.12 Checked on the plates created - no colony growth present. I will attempt to start back at the plasmid and insert preparation step.

10.23.12

By the end of this day I was able to clean up the pcr samples, prepare the plasmid, and transform and plate the bacteria.













10.20.12





Week 7: 102112- Paul, ok - show some data/images. -- Dr. B

10.18.12 Worked on PCR^2 ; I created 16 samples, half for gel extraction and the other half for regular pcr clean up procedure.

10.16.12 continued work on the virtual refresher assignment.

10.15.12 Worked on acquainted self with virtual screening program. Looked over and discussed results of the DNA sequencing and compared it with the online nucleotide BLAST program. Out of the 14 samples sent only 3 had any sort of match up with the gene of interest. Of the three none of the samples had the right size to continue onto the next phase - it can be assumed that only a fragment of the insert actually got into the plasmid. Due to this I will have to restart back at the secondary PCR procedure and try to adjust the protocol to see if theres any change in results.

Week 6: 101612 -Paul - ok good job. Hopefully your cloning works out! - Dr. B 10.13.12

Currently working on virtual screening protocols.

10.12.12 The cleaned up plasmids had concentrations determined through the use of the nanodrop machine. The ranges are from 10-22 ng/ul for plasmid concentration. Concentration seems medium to low. 14 samples were sent to sequencing. 12 were had using the forward primers, and the 2 samples with the highest concentration were used with the reverse primers. Still awaiting results.

























10.9.12 Colonies were grown up in snap tubes and grown up over night. Procedures for the day included spinning down and using the miniprep clean up kit to extract the plasmid.

10.8.12 Plates grew up successfully over the weekend, a nice amount of colonies are present on each of my plates with out too much of a lawn. Colony count seems to be in the 10's. I will proceed to master plating and growing up in tubes.









Week 5: 100912 - Paul - I think that your PCR^2 bands need to be a lot bigger in order to have enough to go on to cloning. Does the gel of PCR^2 correlate to the amounts that you get in Nanodrop. Because the nanodrop vaules seem good for the PCR^2 but the bands are weak. Also, you should have multiple wells of PCR^2 - not just one or two, right? - DR. B Also, try to crop your gels better to get rid of the black space.

10.3.12 ( BSA4 cutting and prepartation - I made 2 preparations of the solution so I could make 2x the plates)







10.2.12 ( I redid my PCR^2 results on gel)





Week 4:100112 - Paul, yeah - make sure there is something there before proceeding. -- Dr. B 9.25.12



Week3: paul - Great. And the 'tail primers' on the left seem to make a larger piece - which is what you want since they should have tails on them! -- Dr. B

Lanes 2 -5 are Max's samples. Both lane 8 and 9 used the oligioprimers and the first and last primers of the deep well box to initiate the pcr reaction. Both were successful in that they had the right size for bp so I will continue on using the oligoprimers as they seem to have a brighter band.

Week2: 9.13.12

9.11.12

Week1:

Due to the change from KOD polymerase to Q5 this week was spent restarting overlaps and transitioning to the new polymerase. My signal in the 4th column for the secondary PCR with Plic forward and reverse primer displayed weak amplification. Possible reasons could be the new polymerase, the slightly altered temperatures for the Q5 thermocycler program affects the primers, or that the primers that I used were old/defective. In any case, since the primary shows a good smear, I'll redo the secondary with the same reagents and find a new plic for/rev sample.

Week 7: After sending dna samples to the lab for sequencing, the results did not match with my target after doing a blast comparison of the sequence. I will restart the experiment from scratch and attempt inserting the target once again.

Week 6: 7.19.12 PCR clean up protocol and concentration by nanospectrometer: Final concentration of plasmid : 26.7 ng/Ul 7. 18.12 Preformed primary and secondary PCR for leishmania major

7.17.12 Worked on primer design and Bsa-4 primer design and found all necessary reagents.

Week 5: Spent the week work on pymol 1,2, and 3. Started reading and looking into Primer design and Bsa-4 primer design

week 4:

7.6.12 Pnic-Bsa 4 PCR results 2



Note: Better results than the first PCR reaction. Bands are not clear/ there is bad resolution. As well there is evidence of PCR in the no DNA control lane. Possible reasons for error: Lanes 2 and 3 could have contaminated lanes 1 and 4. This would be a good reason why the middle 2 lanes have very weak signals while the outer 2 lanes have very strong signals.

7.5.12 Pnic-Bsa 4 PRC results 1

No polymerization chain reaction detected. Possible reasons: The wrong concentration of dNTP was added Wrong primers were added Inadequate amount of DNA was added

7.3.12 PmCherry PCR results
 * [[image:vdsstream/7.3.12 a.JPG width="400" height="300"]]

Note: Unfortunately Taq Pol was left out of the first 4 tubes by accident. To describe the No Dna control of lane 9, it is probably that the results are due to contamination from lane 8.

Week #:

070212 - Paul - good reads on the pNIC_bsa4 sequencing - can you figure out what gene is present in the original vector here? -- Dr. B 6.28.12 Results of DNA sequencing :pNIC-Bsa4 Gene found : <span style="background-color: #ffffff; color: #222222; display: block; font-family: arial,helvetica,clean,sans-serif; text-align: left; vertical-align: baseline;">Bacillus subtilis sacB gene for levansucrase sac B is a counter selection marker. Lavansucrase catalyzes the reaction: sucrose + (2,6-beta-D-fructosyl)n glucose + (2,6-beta-D-fructosyl)n+1

Forward: NNNNNNNNNNNNNNNGGNNNNNNANNNNTGTACTTCCATCNATGGAGACCGACGTCCACATATACCTGCCGTTCACTANN NNNNGTGAAATGAGATATTATGATATTTTCTGAATTGTGATNNNNNNGGCAACTTTATGCCCATGCAACAGAAACTATAA AAAATACAGAGAATGAAAAGAAACAGATAGATTTTTTAGNNNNNNNGGCCCGTAGTCTGCAAATCCTTTTATGATTTTCT ATCAAACAAAAGAGGAAAATAGACCAGTTGCAATCCAAACGAGAGTCTAATAGAATGAGGTCGAAAAGTAAATCGCGCGG GTTTGTTACTGATAAAGCAGGCAAGACCTAAAATGTGTAAAGGGCAAAGTGTATACTTTGGCGTCACCCCTTACATATTT TAGGTCTTTTTTTATTGTGCGTAACTAACTTGCCATCTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTAACACAGTAC ATAAAAAAGGAGACATGAACGATGAACATCAAAAAGTTTGCAAAACAAGCAACAGTATTAACCTTTACTACCGCACTGCT GGCAGGAGGCGCAACTCAAGCGTTTGCGAAAGAAACGAACCAAAAGCCATATAAGGAAACATACGGCATTTCCCATATTA CACGCCATGATATGCTGCAAATCCCTGAACAGCAAAAAAATGAAAAATATAAAGTTCCTGAGTTCGATTCGTCCACAATT AAAAATATCTCTTCTGCAAAAGGCCTGGACNTTTGNGACAGCTGGCCATTACAAAACACTGACNGCACTGTCGCAAACTA TCACGGCTACCACATCGTCTTTGCATTAGCCGGAGATCCTAAAAATGCGGATGACACATCGATTTACATGTTCTATCAAA AAGTCGGCGAAACNTNCTATTGACAGCTGGAAAACGCTNGGCCGCGTCTTTAAAGACAGCGACAAATTCGATGCAAATGA TTCTATCCTAAAGACCAAACNCAANNATNGGTCNGGTTCANCCNCNTTTACATCTGACGGAAAATCCGTTTATTCTACAC TGATTNNTCNGNAAANATTNNGGNAAACNAANNNCTGANANTGCNCAAGNNNNNTATCANCNNCANAANNGCNNTNNNAN NNANGGNGNNNNANGNNNNNNANNANCNTTGNNNGNNNNNGNNAANNTNNNCNAANNNNNNNCNNNNNTCNNNNNANNNC NANNNNCNNNNNNNNNNNNNNNNNNNNNNANNNNNNNNNNNNTNNNNNNAANNANNNNNNAGNNNNNNCNNNNNNNNNN

Reverse: NNNNNNNNNNNNNNNNGNNNNNNGTGNNCGAGTGCGGCCGCAAGCTTGTCGACGGAGCTCGAATTCGGATCCGTATCCAC CTTTACTGGAGACCGTCAATGCCAATAGGATATCGGCATTTTCTTTTGCGTTTTTATTTGTTAACTGTTAATTGTCCTTG TTCAAGGATGCTGTCTTTGACAACAGATGTTTTCTTGCCTTTGATGTTCAGCAGGAAGCTAGGCGCAAACGTTGATTGTT TGTCTGCGTAGAATCCTCTGTTTGTCATATAGCTTGTAATCACGACATTGTTTCCTTTCGCTTGAGGTACAGCGAAGTGT GAGTAAGTAAAGGTTACATCGTTAGGATCAAGATCCATTTTTAACACAAGGCCAGTTTTGTTCAGCGGCTTGTATGGGCC AGTTAAAGAATTAGAAACATAACCAAGCATGTAAATATCGTTAGACGTAATGCCGTCAATCGTCATTTTTGATCCGCGGG AGTCAGTGAACAGGTACCATTTGCCGTTCATTTTAAAGACGTTCGCGCGTTCAATTTCATCTGTTACTGTGTTAGATGCA ATCAGCGGTTTCATCACTTTTTTCAGTGTGTAATCATCGTTTAGCTCAATCATACCGAGAGCGCCGTTTGCTAACTCAGC CGTGCGTTTTTTATCGCTTTGCAGAAGTTTTTGACTTTCTTGACGGAAGAATGATGTGCTTTTGCCATAGTATGCTTTGT TAAATAAAGATTCTTCGCCTTGGTAGCCATCTTCAGTTCCAGTGTTTGCTTCAAATACTAAGTATTTGTGGCCTTTATCT TCTACGTAGTGAGGATCTCTCAGCGTATGGTTGTCGCCTGAGCTGTAGTTGCCTTCATCGATGAACTGCTGTACATTTTG ATACGTTTTTCCGTCACCGTCAAAGATTGATTTATAATCCTCTACACCGTTGATGTTCAAAGAGCTGTCTGATGCTGATA CGTTAACTTGTGCAGTTGTCAGTGTTTGNTTGCCGTAATGTTTACCNGANAAATCANTGTAGAATAAANGGATTTTTCNT CNGANGTNAATGNGNTGNNNGACNTNNNNNNTNNNNNTTTNNNNNNANCATTGCATCNATTGTCNCNNNCTTNNGANNNN NNNNNTTTTNCNNCNNNNANNNANNTNNNNNANTTNNNNNNANNNNNNNNNANNNNNNNCANCNNNTNNNNNNNNNNNNN GNNNNNNNNNNNNNNNNGNNNNNNNNNNNNNNNNN

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6.28.12 Gel of PCR of PBGR22




 * 6.26.12 DNA sequencing results of PMCherry:**
 * 6.26.12 DNA sequencing results of PMCherry:**

Forward: NNNNNNNNCNNNNCGAGGANGNNAACATGGCCNTCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTG AACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGAC CAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACC CCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGC GGCGTGGTGACCGTGACCCAGGACTCCTCCTTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTT CCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCG CCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAG GCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACAC CATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAATAATACTAGA GCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCT CTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGTAGCGGCCGCTGCAGTCCGGCAAA AAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCA CTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTANTACGGTNNTCCACAGAA TCNNGGNANANNCNNNANNANNTGTGAGCAAAAGNCAGCAAAAGNNGNNNTAAAANNNCNTTGCTGNNTTTTCNNNGNTC NCCCCNGANNNNNNTNNNNAAANCGANNNTNANNNNAANGNGNNNNNNNNNNNNNTNNNNNNNNCNGNNTTNCCCNNNNN CNNNNNNNNNNNCNNNNNACNNNNNNNCNNNNNNNNNNNNNTTNNNCNTNGNNANNNNNNNNNNNN

The pmCherry vector tested is indeed mCherry as indicated by a BLAST search.

Cloning vector pME-mCherry-T2A-EGFP, complete sequence Length=4107 Score = 1232 bits (667), Expect = 0.0 **Identities = 678/684 (99%),** Gaps = 0/684 (0%) Strand=Plus/Plus Query 1 AACATGGCCNTCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAAC 60 ||||||||| |||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 698 AACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAAC 757 Query 61 GGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACC 120 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 758 GGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACC 817 Query 121 GCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCT 180 ||||||||||||||||||||||| |||||||||||||||||||||||||||||||||||| Sbjct 818 GCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCT 877 Query 181 CAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTG 240 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 878 CAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTG 937 Query 241 AAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGC 300 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 938 AAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGC 997 Query 301 GTGGTGACCGTGACCCAGGACTCCTCCTTGCAGGACGGCGAGTTCATCTACAAGGTGAAG 360 ||||||||||||||||||||||||||| |||||||||||||||||||||||||||||||| Sbjct 998 GTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAG 1057 Query 361 CTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGG 420 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1058 CTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGG 1117 Query 421 GAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAG 480 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1118 GAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAG 1177 Query 481 AGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCC 540 ||||||||||||||||||||||||||||||||||| |||||||||||||||||||||||| Sbjct 1178 AGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCCGAGGTCAAGACCACCTACAAGGCC 1237 Query 541 AAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCC 600 ||||||||||||||||||||||||||||||||||||||||||||| |||||||||||||| Sbjct 1238 AAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGCTGGACATCACCTCC 1297 Query 601 CACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACC 660 ||||||||||||||||||||||||||||||||||| |||||||||||||||||||||||| Sbjct 1298 CACAACGAGGACTACACCATCGTGGAACAGTACGAGCGCGCCGAGGGCCGCCACTCCACC 1357 Query 661 GGCGGCATGGACGAGCTGTACAAG 684 |||||||||||||||||||||||| Sbjct 1358 GGCGGCATGGACGAGCTGTACAAG 1381


 * Week 2:**

PGFP DH5a E. Coli on Amp. colony plates (6.22.12)-

Colony A - 1 ng/ul ; 1 colony ; 1 efficiency



Colony B - 5 ng/ul ; 544 colonies ; 108.8 efficiency





Colony C - 25 ng/ul of plasmid; 29 colonies ; 1.16 efficiency



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=
================================================== DNA sequencing results ( 6.21.12) pGBR22

Forward:

NNNNNNNNNNNNNNNGNNNNNNNCCGACGTCGCATGCTCCGGCCGCCATGGCCGCGGGATTTTAGTGATGGTGATGGTGA TGACCGAGCAAAGAGTGGCGTGCAATGGATATTTCACACTGCTCAACAAATGTGTAATCCTTGTTGTGACTGGTTACATC CAGTTTGCGGTCAACATAGTGATACCCTGGCATCCTCACAGGCTTCTTTGCCTTGTAAGTAGATTTGAATTCACACAAAT AGTAACCACCTCCTTCCAACTTCAGAGCCATAAAGTTGTTTCCTATCAGCATTCCATCTCGTGCAAAGAGACGCTCAGTG TTGGGTTCCCAGCCCTGTGTCTTCTTCTGCATAACAGGTCCATTGGGAGGAAAGTTCACACCAGAGATTTTGACATTGTA GATGAAACAGTTGCCTTGGATGCTGGAATCATTGCTGACAGTACACACTGCACCATCTTCAAAGTTCATGATCCTCTCCC ATGTATATCCCTCAGGGAATGACTGCTTTACATAATCAGGGATGTCTTCAGGGTACTTGGTGAATGGTATGCTTCCGTAT TGAGACAGTGGTGATAAAATATCCCAAGCAAATGGCAGAGGTCCACCCTTGGTGACAGTGAGCTTTACCGTCTGCTCCCC CTCGTAAGGCTTTCCTTTTCCATCGCCTTCGACCTCAAAGTAGTGTCCATTGACCGTGCCTGACATATAAACCTTGTANG TCATTTGTTTAGCGATCACACTCATGATATTTCTCCTTCAATCAATCAAAATCACTAGTGCGGCCGCCTGCAGGTCGACC ATATGGGAGAGCTCCCAACGCGTTGGATGCATAGCTTGAGTATTCTATAGTGTCACCTNAAATAGCTTGGCGTAATCATG GTCATAGCTGTTTCCNGTGTGAAATTNTTATCCGCTCACAATTCCNCACNACATACNAGCCNGAAGCATAAANNGTAAAG CCTGGGGTGCCTAATGAGTGANNTAACTNNNATTAANTGCNNNGCNCTCACTNCCCGNTTTCCAGTCGGGAAACCTGTCN NGCCNNCTGCATTAATGAATCNGGCNANNNGCGGGNNANNNNNTTNNCGTATGGNNNCNNTTCCGNTNNNNNNNNNNNGN ANTCGNTNNNNNGNNNNTNNNNNNNNNNNNCGGTATNNNNTNNNTNNAGGNGNNNNCNGNNNNNCNNNNNNNNNNNNNNN CCNNNNNANNNNNNNNNNNNNNNCNNNCNANNGNNNNNTNNNNCNNNNNANNNNNNNNNNNNNNNNNNNNNNN

Reverse:

NNNNNNNNNNNNNNANNNTAGNNTACTCAAGCTATGCATCCAACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAGGC GGCCGCACTAGTGATTTTGATTGATTGAAGGAGAAATATCATGAGTGTGATCGCTAAACAAATGACCTACAAGGTTTATA TGTCAGGCACGGTCAATGGACACTACTTTGAGGTCGAAGGCGATGGAAAAGGAAAGCCTTACGAGGGGGAGCAGACGGTA AAGCTCACTGTCACCAAGGGTGGACCTCTGCCATTTGCTTGGGATATTTTATCACCACTGTCTCAATACGGAAGCATACC ATTCACCAAGTACCCTGAAGACATCCCTGATTATGTAAAGCAGTCATTCCCTGAGGGATATACATGGGAGAGGATCATGA ACTTTGAAGATGGTGCAGTGTGTACTGTCAGCAATGATTCCAGCATCCAAGGCAACTGTTTCATCTACAATGTCAAAATC TCTGGTGTGAACTTTCCTCCCAATGGACCTGTTATGCAGAAGAAGACACAGGGCTGGGAACCCAACACTGAGCGTCTCTT TGCACGAGATGGAATGCTGATAGGAAACAACTTTATGGCTCTGAAGTTGGAAGGAGGTGGTTACTATTTGTGTGAATTCA AATCTACTTACAAGGCAAAGAAGCCTGTGAGGATGCCAGGGTATCACTATGTTGACCGCAAACTGGATGTAACCAGTCAC AACAAGGATTACACATTTGTTGAGCAGTGTGAAATATCCATTGCACGCCACTCTTTGCTCGGTCATCACCATCACCATCA CTAAAATCCCGCGGCCATGGCGGCCGGGAGCATGCGACGTCGGGCCCAATTCGCCCTATAGTGAGTCGTATTACAATTCA CTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGNGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTT CGCCAGCTGGCGTAATAGCGAANAGGCCCGCNNCGATCGCCCTTCCNANAGTTGCGCAGCCTGANGGCGAATGGNCNCNN CCNGTAGCGNGCATTNAGCNNNGNGGGNNNNGGNGGNTACNCNCAGCGNNACNCTANNNNNNNGCNCCNNNNNCNNNNNN NTTTNNNTTNNNNNNNNNNNNNCNNNNNNCGNTTCCNNTNANNNNNNNNNGGGGNTNNNNNNNNNATTNNNNNNTNNGNN NNNNNNNNANNTGATNGGNNNNNNNNNNNNNNNNNGNNNNNNNNNNNNNN

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===================================================

Dna extraction of pmCherry Plasmid, nanodrop sample (6.21.12) : 209.9 ng/ul




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