Haynes Lab:Notebook/CRISPR Editing/2016/05/30: Difference between revisions

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<br>
<br>
'''Plate 1'''<br>
'''Plate 1'''<br>
Luc14
:Reps 1-3
::H3K27me3, IgG, input
:::Primer set 1, Primer set 2, Primer set 3
<br>
[[Image:16.05.30 qPCR Plate 1 screen shot.png]]
[[Image:16.05.30 qPCR Plate 1 screen shot.png]]
<br>
<br>
Primer master mix for primer sets 1 and 3<br>
Primer master mix for primer sets 1, 2, and 3<br>
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''1 wells'''
| align="center" style="background:#f0f0f0;"|'''1 wells'''
| align="center" style="background:#f0f0f0;"|'''30'''
| align="center" style="background:#f0f0f0;"|'''34'''
|-
|-
| 2x SYBR mix||7.5||225
| 2x SYBR mix||7.5||255
|-
|-
| 750nmol F/R primer mix||3||90
| 750nmol F/R primer mix||3||102
|-
|-
| PCR water||2.5||75
| PCR water||2.5||85
|-
|-
| total||13||390
| total||13||442
|}
|}


Primer master mix for primer set 2<br>
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''1 wells'''
| align="center" style="background:#f0f0f0;"|'''22'''
|-
| 2x SYBR mix||7.5||165
|-
| 750nmol F/R primer mix||3||66
|-
| PCR water||2.5||55
|-
| total||13||286
|}


<br><br>
<br><br>
Line 66: Line 54:


'''Plate 2'''<br>
'''Plate 2'''<br>
Gal4+dox
:Reps 1-3
::H3K27me3, IgG, input
:::Primer set 1, Primer set 2, Primer set 3
<br>
[[Image:16.05.30 qPCR Plate 2 screen shot.png]]<br>
[[Image:16.05.30 qPCR Plate 2 screen shot.png]]<br>
Primer set 2<br>
Primer master mix for primer sets 1, 2, and 3<br>
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''1 wells'''
| align="center" style="background:#f0f0f0;"|'''1 wells'''
| align="center" style="background:#f0f0f0;"|'''40'''
| align="center" style="background:#f0f0f0;"|'''34'''
|-
|-
| 2x SYBR mix||7.5||300
| 2x SYBR mix||7.5||255
|-
|-
| 750nmol F/R primer mix||3||120
| 750nmol F/R primer mix||3||102
|-
|-
| PCR water||2.5||100
| PCR water||2.5||85
|-
|-
| total||13||520
| total||13||442
|}
|}


<br><br>
'''Plate 3'''
<br>
[[Image:16.05.30 qPCR Plate 3 screen shot.png]]
<br>


Primer set 3<br>
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''1 wells'''
| align="center" style="background:#f0f0f0;"|'''40'''
|-
| 2x SYBR mix||7.5||300
|-
| 750nmol F/R primer mix||3||120
|-
| PCR water||2.5||100
|-
| total||13||520
|}
<br><br>
Make the dilution curves for all three primer sets<br>
Will use just Luc14 gDNA, maybe 10 dilution points in triplicate, will be 90


<br><br>
<br><br>

Revision as of 19:58, 30 May 2016

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05/30/2016

qPCR for ChIP!
Primer sets

Primer set Target F primer R primer amplicon length
1 g025 P331 P329 205
2 g031, g034 P308 P160 202
3 GAPDH P203 P204





Plate 1
Luc14

Reps 1-3
H3K27me3, IgG, input
Primer set 1, Primer set 2, Primer set 3



Primer master mix for primer sets 1, 2, and 3

' 1 wells 34
2x SYBR mix 7.5 255
750nmol F/R primer mix 3 102
PCR water 2.5 85
total 13 442





Plate 2
Gal4+dox

Reps 1-3
H3K27me3, IgG, input
Primer set 1, Primer set 2, Primer set 3



Primer master mix for primer sets 1, 2, and 3

' 1 wells 34
2x SYBR mix 7.5 255
750nmol F/R primer mix 3 102
PCR water 2.5 85
total 13 442




Redo nested PCR of the KAH228/g034 experiments
Did it with the wrong primers the first time, need to redo with P149/176.



Redo nested PCR of the Luc14 g034 samples
Did it with the wrong primers the first time, need to redo with P149/176. (need to double check with notebook to make sure I used 163/215).






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