Template:SBB-PCA Amplification Step
Given to Jon to run program
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.
My sample is the third sample (lane 5)
Ran gel, cut, melted in zymo buffer. Froze before column step.
used PCR tubes to fit in incubator.
only 3 colonies in my plate. Only 2 colonies from class had reasonable results.
Run gel on digest from 3/14/13. With Wells at the top, counting from left to right, lane 3. Gel 2.
Set up new digest from pcr products in tube labeled 3/12/13
started incubation at 10:40
removed from incubator 11:40
zymo cleanup column
1uL Ligation buffer
0.5uL enzyme (last)
1 tube cells
no colonies in new plate, 3 colonies in old plate will be used to proceed.
picked colonies. by ejecting pipet tip into tube.
2) restriction map EcoRI/XhoI
put in thermo cycler 11:23
3) run a gel
Lane 7-Sample A
Lane 8-Sample B
Lane 9-Sample C
4) No results.
31.5 ul H20
10ul Phusion buffer
1ul template (sample from 3/12/13)
1ul 10mm oligo 1 (CCOMT2-3 diluted)
1ul 10mm oligo 2 (CCOMT2-4 diluted)
Tube placed in thermocycler labeled ccomt2 4/16/13
Golden Gate Set up:
(n)ul ddH2O to 10ul
1ul Ligase Buffer
1ul PER CONSTRUCT
sample processed by jc anderson to miniprep step
1ul ligase buffer
1ul of synthon mic