SBB10Ntbk-ChrisAnderson

JCAnderson 13:51, 25 March 2010 (EDT)

I did ca998/g00101 colony pcrs on the Tos clones (16 of them, at far left). I'm miniing and mapping 6, 7, 11, 14. Unfortunately clone 6 didn't grow, so just doing the other 3. Gel is at left, 2nd gel, going 7, 11, then 14 as Eco/Bam digs.

JCAnderson 15:20, 24 March 2010 (EDT)

I mapped CC8, CD8, CG8, and CH8 with Eco/Bam (at right). That's to get more clones of Tos and ZFN. The two ZFN's seem fine. The 2 tos clones are duds. I'll colony pcr more of those.

JCAnderson 16:22, 23 March 2010 (EDT)

The gels above (left is clone 1 , right gel is clone 2). I have 2 more clones of each if this isn't enough. (order of each goes: I, I, D2, E, G, H, R, T, A2, J, K, L, M, N, tos, O)
that maps onto:

sbb35
sbb06
sbb01
sbb20
sbb21
Bca1745
sbb14
sbb04
sbb36
sbb37
sbb39
sbb40
sbb41
sbb03
sbb23

Also, it maps onto staggered lanes from row A and E of "SBB10 Basic Parts C" plate for the first gel, and B and F for the second gel.
During the minipreps, G and H got contaminated. I need to plasmid separate those before a final stock is made of all this.

JCAnderson 16:25, 21 March 2010 (EDT)

Gel at left is the A+B+C SOEing reaction and the D2 SOEing reaction. Both look pretty decent. Gel below that is a repeat of R, S, T pcrs. S, the attP21 site didn't amplify. Need some bonafide MG1655 methinks. The digesting and ligation strategy is here.

JCAnderson 20:00, 20 March 2010 (EDT)

Above right goes A,B,C,D,G,H,L, and M pcrs for the big cleanup set attached here. Those were all 55 pcrs. I cut out A,B,C to cogp. Also cut out D to mix with 2 wobbles.

The gel below that is the 2K55 PCRs going E, F, I, J, K, N. F is phiC31 -- it looks a little off, seems small, perhaps the faint slightly larger band is the right product. Hmm. Ahh crap...construction file is wrong, should be:

 PCR AS009-F/AS012-R on A+B+C	(1868 bp, EcoRI/BamHI)

OK, will redo that tomorrow.

JCAnderson 20:31, 22 February 2010 (EST)

Above right is a gel of 1) sbb24 pcr; 2) MW; 3) sbb15 pcr; 4) sbb31
sbb15 and sbb31 are switched, I switched them back. sbb24 looks fine, I zymo'd it, pdt labeled "sbb24 pcr cleanup". Tubes put in box B.