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Team:British Columbia/Notebook/Labbook
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Revision as of 03:04, 18 October 2014
June - Week 3
June 19th
Experimenter : Dan Korvin, Wenchen Zhao, Zeki Ekmekci
Aim : Amplify T7 RNA polymerase gene (RNAP) from T7 bacteriophage genome and terminator from part BBa_B0015 by PCR.
Result : PCR was successful. Target bands were seen on the agarose gel.
June - Week 4
June 23th
Experimenter : Zeki Ekmekci, Wenchen Zhao, Dan Korvin
Aim : Amplify T7 DNA polymerase (DNAP), primase/helicase (P/H), single strand binding protein (SSBP) genes from T7 bacteriophage genome by PCR. Digest terminator with EcoRI (E) and XbaI (X). Digest RNAP with E and SpeI (S).
Results : NA.
June 24th
Experimenter : Ariel Ragetli, Zeki Ekmekci
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.
Results : DNAP, P/H, SSBP genes successfully amplified, but non-trivial amount of side products (mostly primer-dimer) appeared 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.
Results : 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.
Results : No significant bands we were looking for observed on the gel.
June 30th
Experimenter : Tudor Lapuste
Aim : Amplify four T7 genes and terminator gene by PCR. Digest four genes using E and S.
Results : 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.
Results : PCR failed for both. More PCR set up for primase/helicase.
July 3rd
Experimenter : Ariel Ragetli, Jeffrey Pea, Zeki Ekmekci
Aim : Verify PCR products for P/H on agarose gel. Amplify terminator by gradient PCR. Transform E.coli cells with terminator-containing plasmid.
Results : 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 : Zeki Ekmekci, 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. Transform all four constructs to E.coli
Results : NA
July - Week 3
July 10th
Experimenter : Wenchen Zhao, Zeki Ekmekci
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 : Tudor Lapuste
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 : DNAP and P/H were successfully amplified.
August - Week 1
August 1st
Experimenter : Wenchen Zhao, Dan Korvin, Zeki Ekmekci
Aim : Verify DNAP and P/H constructs by colony PCR. Digest SSBP and RNAP constructs with E and S. Digest the plasmid containing Pbad promoter and RBS with E and X. Transform the ligation products to E.coli.
Results : Colony PCR failed.
August 6th
Experimenter : Wenchen Zhao, Jeffrey Pea, Zeki Ekmekci
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, Zeki Ekmekci
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, Zeki Ekmekci
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 Pbad promoter/RBS found in SSBP and RNAP constructs. Colony PCR result was also negative.
September - Week 1
September 4th
Experimenter : Dan Korvin, Wenchen Zhao
Aim : Double digest DNAP, RNAP and SSBP constructs with X and PstI (P). Ligate three digested genes into RBS + Pbad 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 transform 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 complete DNAP, RNAP and SSBP constructs
Results : NA
September 9th
Experimenter : Wenchen Zhao, Zeki Ekmekci
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 result was negative. No target band was observed. P/H was not mutagenized.
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 result was 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 : Zeki Ekmekci
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 followed by gel check.
Results : Colony PCR result was 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 negative. Pbad romoter/RBS was not present in the sequence. But XbaI restriction site was successfully mutated.
October 2nd
Experimenter : Wenchen Zhao
Aim : Double digest SDM P/H with X and P. Double digest promoter/RBS plasmid with S and P. Ligate digested P/H into digested promoter/RBS plasmid. Transform the ligation product into E.coli.
Results : NA
October 3rd
Experimenter : Wenchen Zhao
Aim : Verify P/H constructs by colony PCR.
Results : Colony PCR result was positive. Target band was observed on the gel.
October - Week 2
October 4th
Experimenter : Ariel Ragetli
Aim : Verify P/H constructs by colony PCR.
Results : Colony PCR result was positive. Target band was observed on the gel.
October 7th
Experimenter : Jeffrey Pea
Aim : Miniprep P/H plasmid and send it for sequencing.
Results : NA
October 8th
Experimenter : Jeffrey Pea
Aim : Double digest P/H with E and X. Double digest 3-gene assembly with E and S. Ligate digested P/H into digested 3-gene assembly. Transform the ligation product to E.coli.
Results : NA
October 9th
Experimenter : Jeffrey Pea
Aim : Verify 4-gene assembly by colony PCR followed by PCR.
Results : Colony PCR result was positive. 4-gene construct was assembled.
October 10th
Experimenter : Wenchen Zhao
Aim : Analyze sequencing result for P/H plasmid.
Results : Sequencing result was negative. There was a ~200bp deletion in the middle of P/H gene. Also there was a 2bp deletion at the beginning of the gene, causing a frameshift mutation
October - Week 3
October 14th
Experimenter : Wenchen Zhao
Aim : Verify protein expression of 4-gene assembly on SDS-PAGE.
Results : DNAP and SSBP protein was successfully expressed. P/H expression was not detected likely due to the mutation. RNAP expression was also not detected.
