Team:British Columbia/Notebook/Labbook

From 2014.igem.org

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               <h1 id="sept_w4"> September - Week 1</h1>
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               <h1 id="sept_w4"> September - Week 4</h1>
               <h3>September 21st</h3>
               <h3>September 21st</h3>
<p>
<p>
Experimenter: Wenchen Zhao
Experimenter: Wenchen Zhao
</p><p>
</p><p>
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Aim: Double digest 3-gene assembly with E and P. Double digest SDM P/H with X and P. Ligate digested P/H to digested RBS plasmid.
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Aim: Double digest 3-gene assembly with E and P. Double digest SDM P/H with X and P. Ligate digested P/H to digested RBS plasmid. Transform ligation product to E.coli.
</p><p>
</p><p>
Results: NA.
Results: NA.
</p>
</p>
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<h3>September 23rd</h3>
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<p>
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Experimenter: Wenchen Zhao
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</p><p>
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Aim: Colony PCR P/H construct.
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</p><p>
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Results: Colony PCR results are positive.
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</p>
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<h3>September 24th</h3>
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<p>
 +
Experimenter: Wenchen Zhao
 +
</p><p>
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Aim: Analyze sequencing results for DNAP, SSBP and RNAP constructs. Miniprep SDM P/H and send it for sequencing.
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</p><p>
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Results: Sequencing results were positive. DNAP, SSBP and RNAP constructs were successfully assembled.
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</p>
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                 <h1 id="oct_w1"> October - Week 1</h1>
                 <h1 id="oct_w1"> October - Week 1</h1>
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               <h3> July 12, 2014 </h3>
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               <h3> October 1st </h3>
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                Sed ut perspiciatis, unde omnis iste natus error sit voluptatem accusantium doloremque laudantium, totam rem aperiam eaque ipsa, quae ab illo inventore veritatis et quasi architecto beatae vitae dicta sunt, explicabo. Nemo enim ipsam voluptatem, quia voluptas sit, aspernatur aut odit aut fugit, sed quia consequuntur magni dolores eos, qui ratione voluptatem sequi nesciunt, neque porro quisquam est, qui dolorem ipsum, quia dolor sit amet consectetur adipisci[ng] velit, sed quia non numquam [do] eius modi tempora inci[di]dunt, ut labore et dolore magnam aliquam quaerat voluptatem. Ut enim ad minima veniam, quis nostrum exercitationem ullam corporis suscipit laboriosam, nisi ut aliquid ex ea commodi consequatur? Quis autem vel eum iure reprehenderit, qui in ea voluptate velit esse, quam nihil molestiae consequatur, vel illum, qui dolorem eum fugiat, quo voluptas nulla pariatur?
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<p>
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[33] At vero eos et accusamus et iusto odio dignissimos ducimus, qui blanditiis praesentium voluptatum deleniti atque corrupti, quos dolores et quas molestias excepturi sint, obcaecati cupiditate non provident, similique sunt in culpa, qui officia deserunt mollitia animi, id est laborum et dolorum fuga. Et harum quidem rerum facilis est et expedita distinctio. Nam libero tempore, cum soluta nobis est eligendi optio, cumque nihil impedit, quo minus id, quod maxime placeat, facere possimus, omnis voluptas assumenda est, omnis dolor repellendus. Temporibus autem quibusdam et aut officiis debitis aut rerum necessitatibus saepe eveniet, ut et voluptates repudiandae sint et molestiae non recusandae. Itaque earum rerum hic tenetur a sapiente delectus, ut aut reiciendis voluptatibus maiores alias consequatur aut perferendis doloribus asperiores repellat…
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Experimenter: Wenchen Zhao
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</p><p>
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Aim: Analyze sequencing results for P/H construct. Digest P/H with E and S and digest 3-gene assembly with E and X. Ligate double digest P/H into double digested 3-gene vector. Transform the ligation product to E.coli.
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</p><p>
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Results: Sequencing result was positive. SDM P/H construct was successfully assembled.
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</p>
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<h3> October 3rd </h3>
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<p>
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Experimenter: Wenchen Zhao
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</p><p>
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Aim: Verify the final 4-gene assembly by colony PCR.
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</p><p>
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Results: Colony PCR result was positive. 4-gene assembly was successfully assembled.
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</p>
           </div>
           </div>

Revision as of 06:59, 17 October 2014

2014 UBC iGEM

Biomining

Orthogonal

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June - Week 3

June 19th

Experimenter: Dan Korvin, Wenchen Zhao

Aim: Amplify T7 RNA polymerase gene (RNAP) and terminator gene by PCR.

Result: PCR was successful. Target bands were seen on the agarose gel.

June - Week 4

June 23th

Experimenter: Wenchen Zhao, Dan Korvin

Aim: Amplify T7 DNA polymerase (DNAP), primase/helicase (P/H), single strand binding protein (SSBP) genes by PCR. Digest terminator with E and X. Digest RNAP with E and S.

Result: NA.

June 24th

Experimenter: Ariel Ragetli

Aim: Verify DNAP, P/H, SSBP genes on agarose gel. Digest DNAP, P/H, SSBP amplicons with E and S. Ligate each digested T7 gene to digested terminator vector.

Result: DNAP, P/H, SSBP genes successfully amplified, but non-trivial amount of side products (mostly primer-dimer) appear in DNAP and P/H lanes. Four T7 genes all digested and ligated with terminator.

June 28th

Experimenter: Wenchen Zhao

Aim: Verify the ligation results on agarose gel.

Result: Multiple bands observed (non-trivial amount of side products). Target SSBP+terminator fragment with distinctive amount observed (only one lane with decent result).

June 29th

Experimenter: Jeffrey Pea

Aim: Amplify the target gene/terminator ligated sequence using four different forward primers and same VF reversed primer. Verify the PCR products on a gel.

Result: No significant bands we were looking for observed on the gel.

June 30th

Experimenter: Wenchen Zhao

Aim: Amplify four T7 genes and terminator gene by PCR. Digest four genes using E and S.

Result: SSBP, DNAP and RNAP were amplified successfully. P/H and terminator PCR failed. PCR for P/H and terminator set up.

July - Week 1

July 2nd

Experimenter: Dan Korvin

Aim: Verify P/H + terminator PCR products on agarose gel.

Result: PCR failed for both. More PCR set up for primase/helicase.

July 3rd

Experimenter: Ariel Ragetli, Jeffrey Pea

Aim: Verify PCR products for P/H on agarose gel. Amplify terminator by gradient PCR. Transform E.coli cells with terminator-containing plasmid.

Result: Terminator and P/H genes amplified.

July 4th

Experimenter: Dan Korvin

Aim: Digest terminator with E and X. Run PCR products of all four T7 genes on a gel, followed by gel extraction and purification.

Results: NA

July - Week 2

July 6th

Experimenter: Wenchen Zhao, Jeffrey Pea

Aim: Double digest all four T7 genes with E and S. Run digested T7 genes and terminator on a gel, followed by gel extraction and purification. Ligate T7 genes with terminator. Transforme all four constructs to E.coli

Results: NA

July - Week 3

July 10th

Experimenter: Wenchen Zhao

Aim: Verify constructs by colony PCR.

Results: Colony PCR failed. No bands were observed.

July 17th

Experimenter: Jeffrey Pea

Aim: Double digest terminator plasmid with E and X. Ligate digested T7 genes to terminator. Transformed four constructs to E.coli

Results: NA

July - Week 4

July 20th

Experimenter: Wenchen Zhao

Aim: Verify constructs by colony PCR.

Results: SSBP and RNAP constructs (gene + terminator) confirmed.

July 24th

Experimenter: Ariel Ragetli, Jeffrey Pea

Aim: Re-amplify DNAP and P/H by PCR followed by gel check. Ligate two digested amplicons to digested terminator. Transform two constructs into E.coli.

Results: NA.

August - Week 1

August 1st

Experimenter: Wenchen Zhao, Dan Korvin

Aim: Verify DNAP and P/H constructs by colony PCR. Digest SSBP and RNAP constructs with E and S. Digest Promoter + RBS plasmid with E and X. Transform the ligation products to E.coli.

Results: Colony PCR failed.

August 6th

Experimenter: Wenchen Zhao, Jeffrey Pea

Aim: Verify DNAP and P/H constructs (gene+terminator), SSBP and RNAP constructs (gene+terminator+RBS+promoter) using colony PCR.

Results: P/H and DNAP constructs confirmed. Bands with correct size were found on the gel after colony PCR of SSBP and RNAP constructs.

August - Week 2

August 12th

Experimenter: Wenchen Zhao

Aim: Prepare SSBP and RNAP miniprep products and send them for sequencing. Double digest P/H and DNAP constructs with E and S. Ligate digested constructs into vector digested with E and X. Transform ligation products into E.coli.

Results: NA.

August - Week 3

August 19th

Experimenter: Ariel Ragetli

Aim: Verify P/H and DNAP constructs using colony PCR. Analyze sequencing results of SSBP and RNAP complete constructs.

Results: Sequencing results are negative – no promoter/RBS found in SSBP and RNAP constructs. Colony PCR results are also negative.

September - Week 1

September 4th

Experimenter: Dan Korvin, Wenchen Zhao

Aim: Double digest DNAP, RNAP and SSBP constructs with X and P. Ligate three digested genes into RBS+Promoter plasmid digested with S and P. Miniprep P/H construct.

Results: NA.

September 5th

Experimenter: Wenchen Zhao

Aim: Transform all three constructs into E.coli. Site-directed mutagenize P/H construct and transforme into E.coli.

Results: NA.

September 6th

Experimenter: Anna Müller

Aim: Verify DNAP, RNAP and SSBP constructs by colony PCR.

Results: Bands with correct size observed, need to send to sequencing for further verification.

September - Week 2

September 8th

Experimenter: Anna Müller

Aim: Miniprep all cultures including SDM P/H construct with terminator and potential complete DNAP, RNAP and SSBP constructs

Results: NA

September 9th

Experimenter: Wenchen Zhao

Aim: Send DNAP, RNAP, SSBP constructs for sequencing. Double digest P/H constructs with X and P, followed by gel check. Double digest RNAP with E and X.

Results: Gel check results are negative.

September 10th

Experimenter: Ariel Ragetli, Jeffrey Pea

Aim: Double digest SSBP with E and S. Double digest RNAP with E and X, followed by gel check and extraction. Ligate digested SSBP into digested RNAP vector. Transform ligation product into E.coli.

Results: NA.

September - Week 3

September 15th

Experimenter: Wenchen Zhao

Aim: Colony PCR SSBP + RNAP construct. Repeat SDM of P/H construct.

Results: Colony PCR failed.

September 16th

Experimenter: Wenchen Zhao

Aim: Re-run colony PCR of SSBP and RNAP construct.

Results: Colony PCR results are positive.

September 17th

Experimenter: Wenchen Zhao

Aim: Miniprep SSBP + RNAP construct, double digest with E and S. Double digest DNAP with E and X, followed by gel verification and extraction. Ligate digested DNAP with digested SSBP + RNAP construct. Transform SDM P/H plasmid.

Results: NA.

September 18th

Experimenter: Wenchen Zhao

Aim: Colony PCR SDM P/H. Double digest P/H with X and P.

Results: Colony PCR failed.

September 19th

Experimenter: Wenchen Zhao

Aim: Repeat colony PCR of SDM P/H. Miniprep DNAP, RNAP for sequencing.

Results: Colony PCR results were positive.

September - Week 4

September 21st

Experimenter: Wenchen Zhao

Aim: Double digest 3-gene assembly with E and P. Double digest SDM P/H with X and P. Ligate digested P/H to digested RBS plasmid. Transform ligation product to E.coli.

Results: NA.

September 23rd

Experimenter: Wenchen Zhao

Aim: Colony PCR P/H construct.

Results: Colony PCR results are positive.

September 24th

Experimenter: Wenchen Zhao

Aim: Analyze sequencing results for DNAP, SSBP and RNAP constructs. Miniprep SDM P/H and send it for sequencing.

Results: Sequencing results were positive. DNAP, SSBP and RNAP constructs were successfully assembled.

October - Week 1

October 1st

Experimenter: Wenchen Zhao

Aim: Analyze sequencing results for P/H construct. Digest P/H with E and S and digest 3-gene assembly with E and X. Ligate double digest P/H into double digested 3-gene vector. Transform the ligation product to E.coli.

Results: Sequencing result was positive. SDM P/H construct was successfully assembled.

October 3rd

Experimenter: Wenchen Zhao

Aim: Verify the final 4-gene assembly by colony PCR.

Results: Colony PCR result was positive. 4-gene assembly was successfully assembled.

© 2014 UBC iGEM

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