Jessica Karen Wong/Notebook/2007-6-27
- PCR E0240
- Colony PCR of Blue C (RBS tester on Chlor)
- Order new T9002, I2055 primers
- Re-transform other devices? Re-ligate?
- Re-PCR I2055 w/ E0240R?
- Did a 100ul preparatory PCR on E0240
- PCR'ed I0255 on a gradient (12 10ul) with the E0240R primer
- Did a colony PCR on the 7 colonies of blue C
- Ran a gel of all PCR products and nothing but the ladders showed up
- Made a new 200nM stock of DNTP's but apparently should have been 2.5 - probably why none of the PCR's worked
- Heat-shocked the digest
- Did a PCR cleanup
- Stored in Measurement Kit box in -20
- Religated B0032-J04650-1AC3 (green C) and B-J-1AT3 (green T)
- Transformed 1ul and plated 100ul of the transformation
cont'd from yesterday
- New T9002 Reverse that matches only GFP TCAGCCAT ATGCAT TTATTATTTGTATAGTTCATCCATGCC
- Melting Temp 55.0
- Original T9002_F CTTAGTAG CAATTG TCCCTATCAGTGATAGAGATTGACATC has melting temp 53.9
Bold is the tail, italics is the restriction site.
- New Longer I2055
- Fwd- CTTAGTAG CAATTG TCCCTATCAGTGATAGAGATTGACATCCCTATC melting temp 62
- Rev- TCAGCGAT ATGCAT TATAAACGCAGAAAGGCCCACCCG melting temp 63.6
- New Shorter I2055
- Fwd- CTTAGTAG CAATTG TCCCTATCAGTGATAGAGATTG melt 45.4
- Rev- TCAGCGAT ATGCAT TATAAACGCAGAAAGGCC melt 45.5
Keep same tails?
- Original I2055
- Fwd- CTTAGTAG CAATTG tccctatcagtgatagagattgacatc melt 53.9
- Rev- TCAGCGAT ATGCAT TATAAACGCAGAAAGGCCCAC melt 53.6 (is actually same primer as E0240R)