SBB10Ntbk-ChrisAnderson: Difference between revisions
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==[[User:JCAnderson|JCAnderson]] 13:51, 25 March 2010 (EDT)== | |||
[[Image:JCA032410gel1.jpg |280px]] | |||
[[Image:JCA032410gel2.jpg |120px]]<br> | |||
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. | |||
==[[User:JCAnderson|JCAnderson]] 15:20, 24 March 2010 (EDT)== | |||
[[Image:JCA032310gel1.jpg |130px|right]]<br> | |||
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. | |||
==[[User:JCAnderson|JCAnderson]] 16:22, 23 March 2010 (EDT)== | |||
[[Image:JCA032210gel1.jpg |300px]][[Image:JCA032210gel2.jpg |300px|]]<br> | |||
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)<br> | |||
that maps onto: | |||
<pre> | |||
sbb35 | |||
sbb06 | |||
sbb01 | |||
sbb20 | |||
sbb21 | |||
Bca1745 | |||
sbb14 | |||
sbb04 | |||
sbb36 | |||
sbb37 | |||
sbb39 | |||
sbb40 | |||
sbb41 | |||
sbb03 | |||
sbb23 | |||
</pre> | |||
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.<br> | |||
During the minipreps, G and H got contaminated. I need to plasmid separate those before a final stock is made of all this. | |||
==[[User:JCAnderson|JCAnderson]] 16:25, 21 March 2010 (EDT)== | |||
[[Image:JCA032110gel1.jpg |100px|left]] | |||
[[Image:JCA032110gel2.jpg |100px|left]] | |||
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 [[Media:032110chart1.xls | here]]. | |||
==[[User:JCAnderson|JCAnderson]] 20:00, 20 March 2010 (EDT)== | |||
[[Image:JCA032010gel1.jpg |200px|right]] | |||
Above right goes A,B,C,D,G,H,L, and M pcrs for the big cleanup set attached [[Media:JCA032010Chart1.xls |here]]. Those were all 55 pcrs. I cut out A,B,C to cogp. Also cut out D to mix with 2 wobbles. | |||
[[Image:JCA032010gel2.jpg |200px|right]] | |||
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. | |||
==[[User:JCAnderson|JCAnderson]] 20:31, 22 February 2010 (EST)== | ==[[User:JCAnderson|JCAnderson]] 20:31, 22 February 2010 (EST)== | ||
[[Image:JCA022210gel1.jpg |200px|right]] | [[Image:JCA022210gel1.jpg |200px|right]] | ||
Above right is a gel of 1) sbb24 pcr; 2) MW; 3) sbb15 pcr; 4) sbb31<br> | Above right is a gel of 1) sbb24 pcr; 2) MW; 3) sbb15 pcr; 4) sbb31<br> | ||
sbb15 and sbb31 are switched, I switched them back. sbb24 looks fine, I zymo'd it, pdt labeled "sbb24 pcr cleanup". | 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. | ||
Latest revision as of 11:41, 25 March 2010
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.
