Haynes Lab:Notebook/CRISPR Editing/2016/02/23: Difference between revisions

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SgrAI destroyed the backbone. Need to cut with more vector, less enzyme and for a shorter time.<br>XbaI cuts in cutsmart, SgrAI cuts in FD buffer.
SgrAI destroyed the backbone. Need to cut with more vector, less enzyme and for a shorter time.<br>XbaI cuts in cutsmart, SgrAI cuts in FD buffer.
<br><br>
<br><br>
'''Western primary stain'''<br>
Make or thaw more blocking buffer (5% BSA, 1%PBS, 1% tween, bring to volume with DI water from tap). Get blocked nitrocellulose membranes from 4deg room.<br>Make parafilm pouches! Cut a strip of parafilm that can be folded over to make a pouch just a few millimeters wider than each membrane. Lay the membrane in the unmade pouch and make the pouch around the membrane by smooshing double-folded parafilm edges with the spatula tool. leave the top open, pipette in 1mL blocking buffer into the pouch, set aside and watch for leaks. Make the other pouches the same way. Dilute the antibodies, 4ul each into 1mL blocking buffer. Confirm that none of the pouches have leaked. Add the antibodies to the appropriate pouches, '''LABEL''' each pouch with the antibody. Seal the tops, rotate at 4degC overnight.
<br>
Antibodies
:H3-ab1791 Rb pAb from abcam
:New - Millipore rabbit poly 07-449 (purchased this month)
:Old - Millipore rabbit poly 07-449 (purchased last year)


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Revision as of 12:02, 27 February 2016

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02/23/2016

Test qPCR primers for ChIP
Dilute Gal4-EED 1 sonicated and column purified DNA from ChIP 1:100
Each reaction

1ul diluted template
1ul F primer
1ul R primer
7ul water
10ul Taq 2x MM


Tm 57

Tube F primer R primer amplicon length Tm for taq
1 P313 P311 186 54
2 P309 P160 171 54
3 P309 P312 175 56
4 P308 P160 202 54
5 P308 P311 171 54
6 P314 P318 205 47
7 P316 P317 169 48
8 P315 P318 197 47
9 P316 P318 160 47
10 P316 P214 196 48



Cut MV13-KAH60
Plasmid: MV13-KAH60 6 297ng/ul, confirmed by Cass
Test SgrAI

1ul SgrAI
1ul Fastdigest buffer or Cutsmart


Test XbaI

1ul XbaI
1ul Cutsmart buffer or Fastdigest

2ul plasmid, 10ul total rxn
Incubate at 37 for 1 hour
Confirm on gel
SgrAI destroyed the backbone. Need to cut with more vector, less enzyme and for a shorter time.
XbaI cuts in cutsmart, SgrAI cuts in FD buffer.

Western primary stain
Make or thaw more blocking buffer (5% BSA, 1%PBS, 1% tween, bring to volume with DI water from tap). Get blocked nitrocellulose membranes from 4deg room.
Make parafilm pouches! Cut a strip of parafilm that can be folded over to make a pouch just a few millimeters wider than each membrane. Lay the membrane in the unmade pouch and make the pouch around the membrane by smooshing double-folded parafilm edges with the spatula tool. leave the top open, pipette in 1mL blocking buffer into the pouch, set aside and watch for leaks. Make the other pouches the same way. Dilute the antibodies, 4ul each into 1mL blocking buffer. Confirm that none of the pouches have leaked. Add the antibodies to the appropriate pouches, LABEL each pouch with the antibody. Seal the tops, rotate at 4degC overnight.
Antibodies

H3-ab1791 Rb pAb from abcam
New - Millipore rabbit poly 07-449 (purchased this month)
Old - Millipore rabbit poly 07-449 (purchased last year)



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