Team:Cambridge/Lab book/Week 9

Monday (20/08/12)

Ratiometrica-Flu: 4th attempt at PCR of fragments

• Only vectors this time as we are still not sure if we have got the Velocity mastermix recipe right, as we are waiting for our orders of Phusion to arrive

• Results: no bands for any lanes including positive control

Ratiometrica-Flu: Restreak of potential Ratiometrica colonies

• One of the 2 colonies from the Gibson reaction and subsequent transformation are restreaked on a Amp 100ug/ml plate

• this is incubated 37°C overnight

Tuesday (21/08/12)

PCR of positive control

• Our order of Phusion has arrived- though we are still not confident with our PCR so we decided to first test with sfGFP

• Standard PCR conditions are used

• Results: success

Ribosense: PCR for Mg Riboswitch

• PCRs were run for pJS130.1 split vector ('+8 codons')
• Colony PCR for endogenous Bacillus subtilis Mg Riboswitch ('+8 codons') was run
• Gels were run for above reactions, only one band for half of the pJS130 vector was visible.

Wednesday (22/08/12)

Ribosense: PCR for Mg Riboswitch

• PCRs were run for F Riboswitch from Yale's plasmid with overhangs for standard assembly.
• Colony PCR for endogenous Bacillus subtilis Mg Riboswitch (both '+8 codons' and '-8 codons' versions) were re-run.
• Gels were run for above reactions, only one band for positive control was visible.

Ribosense: Cells cultured for Miller assay

• B. subtilis strains carrying F riboswitch and 'KO' strains cultured separately in LB and 5µg/mL Chloramphenicol at 37°C
• E.Coli carrying F riboswitch cultured in LB and 50mg/ml Ampicillin at 37°C

Ratiometrica-Lux: PCR of lux-vector

• Subsplit primers for the other half of the fragment that did not come out last week arrived, also new, extended lux FWD primers as we were afraid that the original primers did not anneal properly

• We tried combinations of the new and old primers together with the subsplit and split primers

• Results: only Fragment A2 (Between subsplit FWD and split REV) worked

Ratiometrica-Flu: 5th attempt of PCR of fragments

• Since we got our PCR protocol working, we reattempted the PCR

• Results: the smallest fragments (B0015, K143053) came out, gel extraction is done with the new concentrated protocol

Thursday (23/08/12)

• Miller assay set up and running with the wild type and crcB knock-out Bacillus strains and an E. coli strain which contains the construct on an episomal plasmid.

Friday (24/08/12)

• Miller assay data collected for analysis over the weekend