SBB12Ntbk-DanielChao: Difference between revisions
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No growth in four tubes -> JCA says no red colonies on plate is problem, so redo ligation reaction (may have picked wrong vector digest). So redid ligation reaction -> transformation -> plating. | No growth in four tubes -> JCA says no red colonies on plate is problem, so redo ligation reaction (may have picked wrong vector digest). So redid ligation reaction -> transformation -> plating. | ||
==[[User:Daniel Chao|Daniel]] 12:30, 1 March 2012 (PST)== | |||
Redid A+B SOEing reaction using CA998 + G00101 and A + B fragments. Wait until tomorrow to see results. | |||
Previously plated cells were picked (this time the plate had both red and white cells) and grew in LB tubes. Miniprep was performed on the resulting cell culture: [[Template:SBB-Protocols_Micro3 | Miniprep purification of DNA]]<br>. | |||
The purified DNA was stored. Analytical gel (mapping) will be run next time. | |||
Revision as of 13:07, 1 March 2012
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Daniel 11:30, 16 February 2012 (PST)
Prepared PCR reactions for sb1221:
PCR ca998/gfRevPR on pBjk2741-jtk2801 (160bp, A)
Placed in 100-500 PCR tray
PCR dc5002/g00101 on pBgl00001-Brp0006 (1100bp, B)
Placed in 1K-2K PCR tray
Using protocol as described here: Cloning by PCR
Prepared Wobble reaction for sbb1228
Wobble dc5003/dc5004 (64bp, wobpdt)
Using protocol as described here: Wobble Reaction
Daniel 14:00, 17 February 2012 (PST)
Performed short-fragment Zymo on wobble product, using protocol as described here Small-Frag Zymo Cleanup
Daniel 12:30, 21 February 2012 (PST)
Ran gel of two PCR products used for SOEing Gel pics
Gel #1, lanes 3 and 4 pertain to fragments A (160bp) and B (1100bp) in the construction file for ssb1221.
Cut out gel and purified using Zymo Gel Purification
Performed digest reaction on Zymo wobble product (1 hour in 37C), then purified using Small-Frag Zymo Cleanup.
Daniel 12:30, 23 February 2012 (PST)
Set up final PCR reaction for SOEing.
Performed ligation reaction for sb-1228 Wobble products Ligation of EcoRI/BamHI digests
Heat-shock transformation into cells Transformation by heat-shock
Was not provided with enough cells (only about 100uL in tube). Added 30uL KCM to cells, and used 60uL of mixture for reaction.
Daniel 12:30, 24 February 2012 (PST)
Two things:
1. Picked colonies from plate into four LB tubes for growth.
2. Ran gel on SOEing product to test for product presence, ran out of time to gel purify, so have to wait until next time.
Daniel 12:30, 28 February 2012 (PST)
Ran gel on SOEing product... not sure if products are present.
No growth in four tubes -> JCA says no red colonies on plate is problem, so redo ligation reaction (may have picked wrong vector digest). So redid ligation reaction -> transformation -> plating.
Daniel 12:30, 1 March 2012 (PST)
Redid A+B SOEing reaction using CA998 + G00101 and A + B fragments. Wait until tomorrow to see results.
Previously plated cells were picked (this time the plate had both red and white cells) and grew in LB tubes. Miniprep was performed on the resulting cell culture: Miniprep purification of DNA
.
The purified DNA was stored. Analytical gel (mapping) will be run next time.
