Haynes Lab:Notebook/CRISPR Editing/2016/03/07: Difference between revisions

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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.<br>
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.<br>
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<br>
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<br>
<br>
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
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Tube'''
| align="center" style="background:#f0f0f0;"|'''F primer'''
| align="center" style="background:#f0f0f0;"|'''R primer'''
| align="center" style="background:#f0f0f0;"|'''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).<br>
3. Make primer master mixes for each primer set in separate tubes.
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Reagent'''
| align="center" style="background:#f0f0f0;"|'''Single well'''
| align="center" style="background:#f0f0f0;"|'''x8 (6 dilutions, one blank)'''
|-
| 2x Sybr mm || 7.5 ||60
|-
|750nM primer mix ||3 ||24
|-
|total|| 10.5 ||84
|}
<br>
Prep DNA samples
:Gal4 EED 2, cleaned and sonicated: 204.7 ng/uL
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Dlution'''
| align="center" style="background:#f0f0f0;"|'''Directions'''
| align="center" style="background:#f0f0f0;"|'''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
|}
<br>
for making 1ul per well dilutions for 20 wells<br>
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Reagent'''
| align="center" style="background:#f0f0f0;"|'''Single well'''
| align="center" style="background:#f0f0f0;"|'''11 wells'''
|-
|DNA || 1|| 11
|-
|water ||3.5 ||38.5
|-
|Total ||4.5 ||49.5
|}
<br>
pipet 4.5 ul diluted DNA to each well<br>
pipet 10.5 primer mm to each well




Revision as of 08:41, 7 March 2016

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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



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