Difference between revisions of "User:S Sibert/Notebook/SBB13Ntbk-Stephanie J Sibert"

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This gel is of the PCA2 products for the various synthons. The first lane is the His-tag part's IPCR product (currently unnamed), then molecular weight standards, then the rest are your PCA2's (I couldn't read the labels). All but three look fine. Lanes 4 and 8 look recoverable with a second PCR. The lane 3's reaction needs to be started over from scratch. Lane 7 just needs a careful gel purification.<br>
My sample is the third sample (lane 5)<br>[[Image:2013_03_13-JCA-gel1.jpg]]
Ran gel, cut, melted in zymo buffer. Froze before column step.
Zymo column
ligate [SBB-Protocols_Enz4]<br>
used PCR tubes to fit in incubator.
only 3 colonies in my plate. Only 2 colonies from class had reasonable results. <br>
Run gel on digest from 3/14/13. With Wells at the top, counting from left to right, lane 3. Gel 2. <br>
Set up new digest from pcr products in tube labeled 3/12/13<br>
started incubation at 10:40<br>
removed from incubator 11:40<br>
zymo cleanup column<br>
6.5uL ddH2O
1uL Ligation buffer
1uL vector
1uL insert
0.5uL enzyme (last)
Comp Cells
1 tube cells
30uL KCM
(no ddH2O)
no colonies in new plate, 3 colonies in old plate will be used to proceed.
picked colonies. by ejecting pipet tip into tube.
1)miniprep culture<br>
2) restriction map EcoRI/XhoI<br>
6ul H2O<br>
1ul buffer<br>
2ul DNA<br>
0.5ul XhoI<br>
0.5ul EcoRI<br>
put in thermo cycler 11:23<br>
3) run a gel<br>
Lane 7-Sample A<br>
Lane 8-Sample B<br>
Lane 9-Sample C<br>
4) No results.<br>
Starting with <br>
50ul rxn<br>
31.5 ul H20<br>
5ul dNTP<br>
10ul Phusion buffer<br>
1ul template (sample from 3/12/13)<br>
1ul 10mm oligo 1 (CCOMT2-3 diluted)<br>
1ul 10mm oligo 2 (CCOMT2-4 diluted)<br>
0.5ul Phusion<br>
Tube placed in thermocycler labeled ccomt2 4/16/13
Golden Gate Set up:<br>
(n)ul ddH2O to 10ul<br>
1ul Ligase Buffer<br>
0.5ul Ligase<br>
0.5ul BsmB1<br>
0.5ul vertor<br>
sample processed by jc anderson to miniprep step <br>
sequence confirmed<br>
golden gate<br>
6.5ul ddH2o<br>
1ul ligase buffer<br>
0.5ul ligase<br>
0.5ul BsMb1<br>
1ul of synthon mic<br>
o.5ul vector<br>
1 tube<br>
20ul KCM<br>
no h20<br>
Rescue <br>
6.5ul H20<br>
2ul Plasmid<br>
1ul NEB2<br>
0.5ul Bsa1<br>
lanes 7,8,9 <br>
A,B,D respectivly<br>
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Revision as of 14:20, 1 December 2013

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