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



 * 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