Jessica Karen Wong/Notebook/2007-6-27

To Do

 * 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?

PCR

 * 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

3K3

 * Heat-shocked the digest
 * Did a PCR cleanup
 * Stored in Measurement Kit box in -20

Devices

 * Religated B0032-J04650-1AC3 (green C) and B-J-1AT3 (green T)
 * Transformed 1ul and plated 100ul of the transformation

Designing Primers
cont'd from yesterday

Bold is the tail, italics is the restriction site.
 * 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


 * 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)