Haynes Lab:Notebook/CRISPR Editing/2015/05/27: Difference between revisions

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(Autocreate 2015/05/27 Entry for Haynes_Lab:Notebook/CRISPR_Editing)
 
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==03//2015==
==05/27/2015==
 
Redo qPCR with the right control primers<br>
Dilutions for 2ng gDNA per well:
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Tube label'''
| align="center" style="background:#f0f0f0;"|'''Cell type'''
| align="center" style="background:#f0f0f0;"|'''gRNA'''
| align="center" style="background:#f0f0f0;"|'''plasmid 2'''
| align="center" style="background:#f0f0f0;"|'''ug DNA'''
| align="center" style="background:#f0f0f0;"|'''ul lipo'''
| align="center" style="background:#f0f0f0;"|'''rep'''
| align="center" style="background:#f0f0f0;"|'''ng/µL'''
| align="center" style="background:#f0f0f0;"|'''dilute 1:20'''
| align="center" style="background:#f0f0f0;"|'''ul dilution'''
| align="center" style="background:#f0f0f0;"|'''ul water'''
|-
| 1||Gal4-EED||g034||-||0.5||3||1||81.058||4.05||1.73||14.02
|-
| 2||Gal4-EED||g034||-||0.5||3||2||86.697||4.33||1.61||14.14
|-
| 3||Gal4-EED||g034||-||0.5||5||1||23.89||1.19||5.86||9.89
|-
| 4||Gal4-EED||g034||-||0.5||5||2||24.767||1.24||5.65||10.10
|-
| 5||Gal4-EED||g034||-||1||3||1||85.32||4.27||1.64||14.11
|-
| 6||Gal4-EED||g034||-||1||3||2||70.244||3.51||1.99||13.76
|-
| 7||Gal4-EED||g034||-||1||5||1||13.387||0.67||10.46||5.29
|-
| 8||Gal4-EED||g034||-||1||5||2||23.213||1.16||6.03||9.72
|-
| 9||Luc14||g034||GFP||-||-||1||104.239||5.21||1.34||14.41
|-
| 10||Luc14||g034||GFP||-||-||2||64.965||3.25||2.16||13.59
|-
| 11||Luc14||g034||GFP||-||-||3||39.096||1.95||3.58||12.17
|-
| 12||Gal4-EED+dox||g034||GFP||-||-||1||160||8||0.88||14.88
|-
| 13||Gal4-EED+dox||g034||GFP||-||-||2||129||6.45||1.09||14.66
|-
| 14||Gal4-EED+dox||g034||GFP||-||-||3||132||6.6||1.06||14.69
|-
| 15||water||-||-||-||-||-||-||-||-||15.75
|}


<br><br>
Step 2: make primer master mixes. <br>Will have to add 3.5x to each triplicate gDNA tube so can calculate mastermixes from that<br>
14 samples, 1 tube for no template control, total of 15 tubes per primer.
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''1rxn'''
| align="center" style="background:#f0f0f0;"|'''3.5/triplicate tube'''
| align="center" style="background:#f0f0f0;"|'''15.7'''
|-
| primer||3||10.5||164.85
|-
| SYBR||7.5||26.25||412.125
|-
| Total||10.5||36.75||576.975
|}
Make these mixes for each primer pair, add 36.75 to each triplicate tube, then pipette 15ul into 3 well of the plate.


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Revision as of 17:18, 27 May 2015

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05/27/2015

Redo qPCR with the right control primers
Dilutions for 2ng gDNA per well:

Tube label Cell type gRNA plasmid 2 ug DNA ul lipo rep ng/µL dilute 1:20 ul dilution ul water
1 Gal4-EED g034 - 0.5 3 1 81.058 4.05 1.73 14.02
2 Gal4-EED g034 - 0.5 3 2 86.697 4.33 1.61 14.14
3 Gal4-EED g034 - 0.5 5 1 23.89 1.19 5.86 9.89
4 Gal4-EED g034 - 0.5 5 2 24.767 1.24 5.65 10.10
5 Gal4-EED g034 - 1 3 1 85.32 4.27 1.64 14.11
6 Gal4-EED g034 - 1 3 2 70.244 3.51 1.99 13.76
7 Gal4-EED g034 - 1 5 1 13.387 0.67 10.46 5.29
8 Gal4-EED g034 - 1 5 2 23.213 1.16 6.03 9.72
9 Luc14 g034 GFP - - 1 104.239 5.21 1.34 14.41
10 Luc14 g034 GFP - - 2 64.965 3.25 2.16 13.59
11 Luc14 g034 GFP - - 3 39.096 1.95 3.58 12.17
12 Gal4-EED+dox g034 GFP - - 1 160 8 0.88 14.88
13 Gal4-EED+dox g034 GFP - - 2 129 6.45 1.09 14.66
14 Gal4-EED+dox g034 GFP - - 3 132 6.6 1.06 14.69
15 water - - - - - - - - 15.75



Step 2: make primer master mixes.
Will have to add 3.5x to each triplicate gDNA tube so can calculate mastermixes from that
14 samples, 1 tube for no template control, total of 15 tubes per primer.

' 1rxn 3.5/triplicate tube 15.7
primer 3 10.5 164.85
SYBR 7.5 26.25 412.125
Total 10.5 36.75 576.975

Make these mixes for each primer pair, add 36.75 to each triplicate tube, then pipette 15ul into 3 well of the plate.


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