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Team:British Columbia/Notebook/Labbook
From 2014.igem.org
June - Week 3
June 19th, 2014
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, 2014
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, 2014
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, 2014
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, 2014
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, 2014
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, 2014
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, 2014
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, 2014
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, 2014
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, 2014
Experimenter: Wenchen Zhao
Aim: Verify constructs by colony PCR.
Results: Colony PCR failed. No bands were observed.
July 17th, 2014
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, 2014
Experimenter: Wenchen Zhao
Aim: Verify constructs by colony PCR.
Results: SSBP and RNAP constructs (gene + terminator) confirmed.
July 24th, 2014
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, 2014
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, 2014
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, 2014
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, 2014
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, 2014
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, 2014
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, 2014
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 9th, 2014
September - Week 3
July 12, 2014
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July 12, 2014
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July 12, 2014
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