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) 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.

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: Tudor Lapuste

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. Transform 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: 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

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 negative. Promoter/RBS was not present in the sequence. But XbaI restriction site was successfully mutated.

October 3rd

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 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.