ResearchPage+-+Shreya

Shreya's Research
Week 12

- Completed Protein purification - Completed the nanodrop for the Elution 1 and 2 which are shown in the following picture:

Elution 1 for Carbonic Anhydrase

Elution 2 for Carbonic Anhydrase

- Continued working on Virtual Screening

Week 11

- Attempting to complete virtual screening for Carbonic Anhydrase ( Completed the 2nd run of the HF9 Library) - Completed material and methods for virtual screening - Completed the 4th day of protein expression. Stored in -4 degrees Celsius Fridge.

Proposed Goals:

- Check to see if my procedure worked for protein expression - If not, repeat the procedure.

Week 10

After a few attempts on cloning, skipped onto protein expression ( completed till day 4). Completed the steps needed. Weighted the pellet at 3.38 grams.

- Continued Virtual Screening for Carbonic Anhydrase

Proposed goals for this week:

Complete Virtual Screening for Carbonic Anhydrase - Continue Protocol for protein expression

Lastly completed the material and methods procedure for cloning- sent email to Dr. B with assignment.

Week 9

- Virtual Screening did not work for proposed target. Completed another run on October 25, 2011. Complete run this week.

The following goals are proposed:

- Complete PCR overlap - Complete the Virtual Screening for carbonic anhydrase

Long term goal: Start cloning of the gene

Week 8

Completed the virtual screening of my protein ( carbonic anhydrase). As of now, the information is on queue.

Virtual Refresher- completed on Tuesday- due to the break in the blades- rerun is needed

Next step complete the PCR overlap. - Completed PCR overlap- however, cannot find my product in fridge. Re- do is needed

Week 7 Shreya - is this Week 7? Break up each by week. -- Dr. B The results for the PCR of the p-Lic-BSa4 are shown in the following picture:



The following picture shows Tube A- Tube D for pLic PCR. None of the samples showed up for the following PCR. Only the 1kb or 100 bp ladder showed up.

The results for the RE digest are shown in the following image:



After letting the sample sit in Ethidium bromide overnight- the following results showed up: Sample 1 ( Contains the EcoRI), 2 ( Contains the PvuII), 3 ( EcoRI+ PvuII) are in lanes 4-6. With 2 micro liters of the blue juice added to each sample.

Protocol added to notebook as well as the results.

The virtual screening started- information out of queue. . . . started running this Friday.

Week 6

PCR Primer Design for Primer Overlap Assembly PCR

The sequence is for Ecoli Class II.

1 ATGAAAGACATCGACACCCTGATCTCTAACAACGCGCTGTGGTCTAAGATGCTGGTT 57 2 CGCCTGCGCCAGTTTTTCGAAGAAACCCGGATCTTCTTCAACCAGCATCTTAGACCACAG 60 3 AAAAACTGGCGCAGGCGCAGAAACCGCGCTTCCTGTGGATCGGTTGCAGCGACTCTCGCG 60 4 GAACAGTTCACCCGGTTCCAGACCCGTCAGACGTTCCGCCGGTACGCGAGAGTCGCTGCA 60 5 GGAACCGGGTGAACTGTTCGTTCATCGTAATGTTGCGAACCTGGTTATCCACACCGACCT 60 6 CCAGAACATCAACAGCGTACTGAACAACAGACAGGCAGTTCAGGTCGGTGTGGATAACCA 60 7 AGTACGCTGTTGATGTTCTGGAAGTTGAACACATCATCATCTGCGGTCATTCTGGCTGCG 60 8 ATCAGACCCAGTTCCGGATTTTCAACCGCCGCTTTGATACCACCGCAGCCAGAATGACCG 60 9 ATCCGGAACTGGGTCTGATCAACAATTGGCTGCTGCACATCCGTGACATTTGGCTCAAAC 60 10 AGGCGCTGTTCCTCCGGCATTTTACCCAGCAGGGAAGAGTGTTTGAGCCAAATGTCACGG 60 11 CGGAGGAACAGCGCCTGGATGCGCTCTACGAACTGAACGTTATGGAGCAGGTTTACAACC 60 12 ACGTTTCCACGCAGACTGCATGATGGTAGAGTGACCCAGGTTGTAAACCTGCTCCATAAC 60 13 CAGTCTGCGTGGAAACGTGGTCAGAACGTCACCATCCACGGTTGGGCGTACTCTATCAAC 60 14 GTTAGTCGCGGTAACGTCCAGGTCACGCAGCAGACCGTCGTTGATAGAGTACGCCCAACC 60 15 GGACGTTACCGCGACTAACCGTGAAACCCTCGAAAACGGTTACCATAAAGGCATCTCTGC 60 16 TTACTGGTGCGGGATGTATTTCAGAGACAGCGCAGAGATGCCTTTATGGTAAC 53

Completed the second PCR primer design for pNIC-BSa4 Cloning.

Placed both documents- in google docs- under my folder "Shreya Narang."

Plans for next week

Complete the PCR overlap

Complete the virtual screening ( As it just started running yesterday)

Week 5 October 7,2011

For the ethidium bromide soaking did work for the Re Digest in the previous week.

Image in spiral.

The following the week the following goals are proposed

1.) Virtual Screening Refresher Completed first part of the refresher process- but because the blades are full on the database information is in queue . . . continue to run until put on blade

2.) PCR for the P-Lic

Completed- place in 4 degrees Celsius Fridge Run the gel- electropheresis for Monday.

3.) PCR overlap

Part completed- placed in 4 degrees Celsius Fridge Run the gel- electropheresis

3.) Primer design- To be completed next week

Week 5 September 23, 2011 - Shreya - I am glad the Ethidium Bromide Soaking worked - is this the gel for that or another one? - Dr. B

For the following week the following gains were attained:

1.) Completed 2nd PCR of PGBR22

Protocol for this PCR mentioned previously.



Lane 1 was left blank. 100 BP ladder was shown up in the 2nd lane. Tube A with the 0.3 nanograms of PGBR22 is shown in Lane 3. Tube B with 3.0 nanograms of PGBR22 is shown in Lane 4. Tube C with the 30 nanograms of PGBR22 is shown in Lane 5. Tube D with no PGBR22 (or the control) is shown in Lane 6.

Re-digest completed- ran the gel however the gel was no running. Added 1 microliter of ethidium bromide, waited 30 minutes to see whether the gel will run. (Trial and error) Bands did show up after 30 minutes, two additional microliters of ethidium bromide were added. ( Leaving overnight)

2nd PCR of pNIC28-Bsa4- completed Run the gel for next Monday

In addition the pymol refresher was completed this week.

Week 4

For the following past weeks the following goals were attained:

1st week 1.) Completed the "analyzing DNA sequence assignment." The link is posted below as well as updated on google docs:



or under google docs " san587_ShreyaNarang_Analyzing DNA sequence_ 9/6/2011"

2nd week

For the PCR

1st step: Dilute the template 2nd step: Create the Master Mix 3rd step: Add the Master mix to each tube 4th step: Add template to each tube 5th step: Add water to each tube 6th step: Place the tubes on the ice 7th step: Reserve a PCR machine and breheat to 94 degreees Celsius and label with tape ( Name, Date) 8th step: Dilute Taq(Enzyme sensitive to the heat) 9th step: Add the Taq 10th step: Transfer the reaction to the thermocycler preheated to denaturation (94 degrees Celsius) 11th step: Run Thermal Program

2nd part of the PCR

1.) Create Agarose Gel 2.) Run through electrophoresis

Completed the first PCR for the PGBR22 using SP6. The results were inconclusive.( See picture below)

Document posted on google docs: "san587_ShreyaNarang_PCR#1forPGBR22"



Lane 1 of the PCR was skipped, lane 2 was the DNA ladder (1KB), lanes 6-10 were also DNA ladders ( for practice), lanes 3-6 were the PCR PGBR22.

3rd Week

1.) Re-did PCR using the M13 instead of SP6 with some modifications in the calculations ( as shown in laboratory notebook). Placed in thermocycler to run throughout the program.

Plans for tomorrow: Run the gel through electrophoresis; possibly start RE digest procedure (Friday/Monday)

2.) 3 out of the 4 Pymol assignments were completed this week.



SN1 DNA sequencing using the SP6 primer NNNNNNNNNNNNNNGGNNANTGNGGNNCNANNTCGNCATGCTCNCGGCCGNCNTGGCCNNNNGATTTTCGTGATGGTGATGGTGATGACCNANNAAAGAGGGGGNTGAAATGGATATTTNNCACTGCTCAACAAATGTGTAATCCTTGTTGTGACTGGTTACTTCCAGTTTGCGGTCGACATATTGATACCCTGGCATCCTCACAGGCTTCTTTGCCTTGTAAGTAGATTTNATTTCACACNAATAGTANCCNCCTCCTTCCAACTTCANAGCCATAANGNTGTTTCCTATCNNCATTCCATCTCTCGCAAAGAGACTCTCACTGTTGGGTTCCCANCCCTGTGTCTTCTTCTGCATAACAGGGCCTTTGGGAGGAAAGTTCGCACCNNAGATTTTGACTTTGTACATGAAACTTTTGCCTTGGATGCTGGAATCATTGCTGACAGGACACACTGCACCNTCTTCNAAGTTCCTGATCCTCNNNCATGTNTNNNNNTCNNGGAANGANTGCTTTCCTTANGCAAGGATGNCTTTNNGGTACTTGGNNANTGGTATGCTTCCNTNTTGANNCNNNGGNGATAAAATATCCNCAGCAANTGGNANATGTCCNCCCTTGGTGACAGAGANCTTTACCNNCTGCTCCCCCTCTTAAGGNTNTCCTTTTCCNTCNCCNGCTACCTCNAANTATTGNCCATTGACNNTGCCTGACNTNNANNNNNNGTANGTCATTTGTTNANCNATCACACTCNTGATATNTCTCCTTCACTCNNNNNANATCTCTANTGCGGNCNCCTGCNGGTCGANNNTATNGNAGAGNTNCCNACCCGTTGGATGCNTACCTTGAGTATTCTATANTGNCACNNNAATANCTTGNCGTATCATNNTCATANCTGNNTCCTGTGNGAAATTGNTATCNNNCTCNCANNTNCTCACAACNTACTNANCCTGAANCATAAANTGNAAAGCCNGNNGNTGNCNNATNANNNNNNNNNCTNNCNTNNNTTGNNTNNCNNCTCNCTNNNCTNNTTTCNNNCNNNNNNNNNGNNNTGNCNNCNGNANNNATGNNTCGNCNACTGCNNNNAGNANNNNNNNNNNNNCNNNNGGNNNNNTNNTNCNNNTTCNTNGNTNNNNNGANNCNCTNNNNTCGTNNNNNNNNTNNNNNNNNNGNNTNNGNTNNNNNNNANGNNGNNNNNNNNNNTNTNCNANNNNNNNNNNNNNNNNNCNNGNANNNNNNNNNNNCNNNNNNNNNANNGCNNNNNNNNAANANNNNNNNNNNNNNNNNNNNNNNNNNNCNNNNNGGNNN

Blast Sequencing for SN1 sequence



Nucleotide Sequence



SN2 DNA sequencing using the T7 primer NNNNNNNNNNNNNNNGNNNNNGNNNNCTGNCCNCNGGNNTNANCTGNNGGNGNNNGCGATNANGNATNNNATTGTGTGAAGGAGNGANNATTTGANTGNNTCNCTAATGTGTGACCTNGTTGTGANTGGGTANNGCNGGTTGCGGGNCNCTANNNNNANGCNGAANGNNTNGAGGCTTCNTTGCCTTGAGGGGGATTTNANTTNNNANNTCACTGTCCCNCNGGNGGACCNNTNCCANTTGCTTGGGAGTTNCNNNTCNACNGNNNNCTACNGAAANAGACGCTCACNGTTGGGTTCCCNNACCTGTGTGATCTTCTGAANNANTCGTTCNNTGNGGGAAAAGTTCGGNANANNNTTNTGAACTTTGAAGATGAAACAGTNNCNNNGGATGCTGAAAACTTTGNNAACNNNNGNNACTGNNNCATCTNCAANGTNNANGATNCTNGNGNNNGNNTNNCCCNCAAGGAANGNGTGNTTNANAAAAACANGNANGNCTNNNNGNNCCNNGNTGAANGNNNNNNTNCCNNANANNGANNGNNGNGANAAAANANCNNNANNGNATGGAANANGNNNNGNNGTGGTNACNATNNGTNTNACNNNNNGNNCNCNNNCNAGGNGNNNNNNNTTGNNANGATGCNNGNNNATCACTTNGTGNACNTTGANCNGGACNNANNCANNCACNNTGNNGATNANNNGTTTNNNGATCACNGTNANNNATANNTNTGCNNCNNNCTCTCTGNNTCNCCATCGCCNTCNCCNTGCACTGTCNACCNGNGGNNATANCNNCCNGGAGCNTGNANNCNTNGNNNNANTNTNNCNNTATNGTGACNTCANATNNCNNGTCNNNNNCNTGNNNNTNNCTGCNTCNTGACTGNNANTGNNNTNNNNTNACNNTCCNNANNNNNTANNNGCNNNANNCNNNTNNGCANNNNGNNNNGTGCCNANAGNNAGCNCCNTNNNNNATNNNANNNTGNNNNNNNNCNNANTNNCNANNNNGANNCTNNNNGNNNCNTNATNNNNGNNNNGGGNNNNNNGNTNNGGNNNANNNNNNNNNNNNANTNGGNNNNNNNNNNNNNNNNNNNNNNNNTNNTNNNNNNNNNNGNNNNNNNNNGTNNCCNNNANCNNNNNNNNNNNNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGNNNNNNNNNNNNNNNNNNNNNCNNNNNNCNANNNNNNN

Blast Sequencing using the T7 primer

I'm not getting any similar blast sequencing using the T7 primer.

Nucleotide Sequence using the T7 primer

Objectives for next week

- Complete RE Digest