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

• 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)