03/07/2016
PCR of gB009 for cloning EGFP behind dCas9. Four reactions of 100ul each, 1:100 dilution (0.2ng/ul) gBlock, 1ul P319/320, gotaq 2x mm.
added 1mL media to the Friday transfections before 7am, will collect around noon
qPCR to test primers for ChIP. From the first run on http://openwetware.org/wiki/Haynes_Lab:Notebook/CRISPR_Editing/2016/02/23, we know that only P316/214 give second product with Taq.
DNA template is Gal4-EED sonicated (2) and cleaned up DNA from http://openwetware.org/wiki/Haynes_Lab:Notebook/CRISPR_Editing/2016/02/05#02.2F05.2F2016
1. Make ~750 nM F/R primer mixes: for each primer set, add 3.8 μLof 100 μM forward primer and 3.8 μL of 100 μM reverse primer to 492.4 μL H2O
| Tube
|
F primer
|
R primer
|
amplicon length
|
| 1 |
P313 |
P311 |
186
|
| 2 |
P309 |
P160 |
171
|
| 3 |
P309 |
P312 |
175
|
| 4 |
P308 |
P160 |
202
|
| 5 |
P308 |
P311 |
171
|
| 6 |
P314 |
P318 |
205
|
| 7 |
P316 |
P317 |
169
|
| 8 |
P315 |
P318 |
197
|
| 9 |
P316 |
P318 |
160
|
2. Label one 1.5 mL tube per dilution (final volume = 500 μL).
3. Make primer master mixes for each primer set in separate tubes.
| Reagent
|
Single well
|
x8 (6 dilutions, one blank)
|
| 2x Sybr mm |
7.5 |
60
|
| 750nM primer mix |
3 |
24
|
| total |
10.5 |
84
|
Prep DNA samples
- Gal4 EED 2, cleaned and sonicated: 204.7 ng/uL
| Dlution
|
Directions
|
Final conc ng/ul
|
| 1:10 |
2 into 18 |
20.47
|
| 1:50 |
4 of 1:10 into 16 |
4.094
|
| 1:100 |
2 of 1:10 into 18 |
2.047
|
| 1:500 |
4 of 1:100 into 16 |
0.4094
|
| 1:1000 |
2 of 1:100 into 18 |
0.2047
|
| 1:5000 |
4 of 1:1000 into 16 |
0.04094
|
for making 1ul per well dilutions for 20 wells
| Reagent
|
Single well
|
11 wells
|
| DNA |
1 |
11
|
| water |
3.5 |
38.5
|
| Total |
4.5 |
49.5
|
pipet 4.5 ul diluted DNA to each well
pipet 10.5 primer mm to each well
|
Project name
Main project page
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