Gary Dixon 13:06, 10 March 2011 (EST)
Loading the best clones into the plates for sequencing. Clones chosen: 3 and 4 of sbb1117 (loaded into B1 of each plate). 2 and 3 of sbb1127 (loaded into A1 of each plate).
Gary Dixon 12:51, 8 March 2011 (EST)
Doing mapping today.
Have eight minipreps, so created a master mix of the water, buffer, enzymes:
8uL 10x NEB Buffer 2
Then separated into eight small tubes with 6ul of mix per tube.
Then added 4uL Miniprepped plasmid to each tube.
Then continued with the procedure:
* Incubate at 37 on the thermocycler for 30 minutes * Run an analytical gel * Take a picture of the gel * Calculate the expected fragment sizes * Are the calculated sizes consistent with the bands on the gel?
Gary Dixon 14:42, 3 March 2011 (EST)
The plates looked good. Lots of big colonies on both of them. Only ~5 redish colonies on each. The sbb1117 plate had about 1/3 more colonies than the sbb1127 plate.
The powers at be picked the colonies and inoculated for us. 8 colonies were inoculated (2 for each part).
Miniprep Procedure: http://openwetware.org/wiki/Template:SBB-Protocols_Micro3
There were 8 products in all labeled: 1117 1:MP, 1117 2:MP, 1117 3:MP, 1117 4:MP, 1127 1:MP, 1127 2:MP, 1127 3:MP, 1127 4:MP
Gary Dixon 12:44, 3 March 2011 (EST)
FEB 24th did not get posted for some reason... I must not have saved.
That was the day I did digestion/clean-up of the SOEing product to get ready for ligation.
Gary Dixon 15:26, 1 March 2011 (EST)
Performed two ligation reactions for the two parts and their vectors: http://openwetware.org/wiki/Template:SBB-Protocols_Enz4.
Then performed two transformation reactions using Lefty Comp Cells: http://openwetware.org/wiki/Template:SBB-Protocols_Micro1
The water and KCM were not added to the cells until after the 30min incubation step (in the ligation reaction) was finished.
I let the ligation reactions cool on ice for 2 min before adding the competent cells.
Entered the shaker at 11:10am.
Taken from the shaker at 12:10pm.
Plated on Cam plates labeled: Gary Cam. sbb1117 and Gary Cam. sb1127
Put to the side for overnight incubation.
Gary Dixon 15:13, 22 February 2011 (EST)
Used the wrong Wash buffer for zymo cleanups, so Professor Anderson redid the PCRs.
Ran another analytical gel for SOEing product sbb1127.
Performed another Zymo cleanup on sbb1127. Did not start digestion because running low on time.
EcoRI/BamHI digest of Part 1117: http://openwetware.org/wiki/Template:SBB-Protocols_Enz2
Entered thermocycler at 10:24. Removed for Prep gel at 11:25.
Prep gel started at 11:43.
Cut out after class by Professor Anderson. Thanks Professor!
Gary Dixon 16:36, 18 February 2011 (EST)
Ran an analytical gel for the SOEing PCR product that used 53/53 oligos.
Gel confirmed that the major PCR product is around 981bp. There is another band that is not the desired product. It looks like it is around 300bp, which suggests that is a result of the B template from the first PCR reaction. There will be a round of gel purification after digestion, so they will be separated.
Proceeded to clean up the PCR product using the Regular Zymo Cleanup Protocol: http://openwetware.org/wiki/Template:SBB-Protocols_Zymo1
Put into box A labeled as GD 53/53 PCRP
Gary Dixon 13:22, 17 February 2011 (EST)
Running two preparative gels:
53/31 PCR = GD1 (#3)
32/53 PCR = GD2 (#4)
5ul of product
5ul of loading dye
And one analytical gel:
42/42= GD3 (#6)
2ul of product
5ul of loading dye
Both gels looked good.
Ran preparative gel, did surgery, and melted the gel with 600ul of ADB buffer.
Did regular Zymo clean-up for 42/42. Storing it in box as GD 42/42 PCRP.
Did Zymo gel purification for 32/53 combined with 53/31.
Used the product of the Zymo gel purification as the template for the PCR with both 53 oligos. Placed in <2kb PCR because PCR product should be 981bp.
Gary Dixon 14:32, 15 February 2011 (EST)
Performing 3 PCR reactions. One for part sbb1117, and two for part sbb1127 (soeing).
First, sorted out some confusion about letter R and F on oligo. R and F don't necessarily correspond to your forward and reverse oligo. So ss53r is the forward oligo for P_nmpC and ss53f is the reverse.
Then diluted oligos gd031 and gd032.
Then went through the standard procedure to set up PCR reactions. I have three in total.
ss53r/gd031R (718bp, gp = A)
gd032F/ss53f (287bp, gp = B)
The procedure: http://openwetware.org/wiki/Template:SBB-Protocols_PCR2
Put all three PCR reactions in the <2,000kb reaction.
Gary Dixon 23:59, 5 February 2011 (EST)