Jessica Karen Wong/Notebook/2007-6-22

To Do

• Run gel of T9002 and E0240 PCR
• PCR Cleanup of T & E
• Digest T & E with Mfe1 and Nsi1
• PCR cleanup B, J, and R
• Ligate and transform B, J, R
• Log outline, schedule, and parts

Digest Cont'd

• heat shocked digests for J04650, B0032, R0040, and the plasmids
• put in 4 degree

Gel

Gel from 6/22/07

• Ran an analytic gel of the PCR product of T9002 and E0240
• L to R: Ladder, E, T, Ladder, space, Ladder
• E0240 gave no results and the T9002 fragments are 1kb when the desired product should have been 2kb
• T9002 has to B0015 sites in it and we conclude it must have cut only at the first site
• This also explains the absence of E0240 b/c they have the same end sequences
• Ordered new reverse, E0240F, and T9002F primers

Designing Primers

• New Reverse Primer (both T9002 and E0240) TCAGCCAT ATGCAT AAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGATGCCTGGCTCTAGTATTATTATTTG
  • Melting temp without tail is 81.5
  • Is so long because it must go all the way back to some of the GFP

New forward E0240 CTTAGTAG CAATTG TCACACAGGAAAGTACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAG

• • Melting temp without tail is 80.9
  • Length is to have similar melting temp as reverse

New forward T9002 CTTAGTAG CAATTG TCCCTATCAGTGATAGAGATTGACATCCCTATCAGTGATAGAGATACTGAGCACTACTAGAGAAAGAGGAGAAATACTAGATG

• • Melting temp 80.0

Bold is the tail, italics is the restriction site.

PCR Purification

Did a PCR cleanup of the 3 backbones, T9002, E0240, B0032, R0040, and J04650

• Used 50ul of B, R, and J
• Used 90ul of T & E
• Protocol is in QIAquick spin handbook

3-way Ligation

Nishant Ligated:

• R0040, J04650, and 1AT3
• R, J, and 1AC3
• B0032, J04650, and 1AT3
• B, J, 1AC3

Overnights

• Checked competent cells by transforming with pUC18 and plating on both plain LB and Amp
• Set up 2 5ml overnight cultures of each plasmid (1AT3, 1AC3, 1AK3, and 3K3)
  • 1 to miniprep and 1 to make glycerol stocks