Haynes Lab:Notebook/CRISPR Editing/2015/05/22: Difference between revisions

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(Autocreate 2015/05/22 Entry for Haynes_Lab:Notebook/CRISPR_Editing)
 
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==03//2015==
==05/22/2015==
'''SYBR qPCR to determine plasmid counts for dilution experiment'''
{| {{table}}
| pX330 primer||Primer G
|-
| internal primer||Primer C
|-
| Dilution||2ng/rxn
|}
''Step 1: make two PCR tubes for each sample with the following volumes of water and gDNA for 2ng of gDNA per well''
:{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''1rxn'''
| align="center" style="background:#f0f0f0;"|'''x3.5 = 7ng'''
|-
| gDNA||x||
|-
| water||to 4.5||
|-
| Total||4.5||15.75
|}
:{| {{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;"|'''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 1:10'''
| align="center" style="background:#f0f0f0;"|'''ul dilution'''
| align="center" style="background:#f0f0f0;"|'''ul water'''
|-
| 1||Gal4-EED||g034||0.5||3||1||81.058||4.0529||1.73||14.02
|-
| 2||Gal4-EED||g034||0.5||3||2||86.697||4.33485||1.61||14.14
|-
| 3||Gal4-EED||g034||0.5||5||1||23.89||1.1945||5.86||9.89
|-
| 4||Gal4-EED||g034||0.5||5||2||24.767||1.23835||5.65||10.10
|-
| 5||Gal4-EED||g034||1||3||1||85.32||4.266||1.64||14.11
|-
| 6||Gal4-EED||g034||1||3||2||70.244||3.5122||1.99||13.76
|-
| 7||Gal4-EED||g034||1||5||1||13.387||0.66935||10.46||5.29
|-
| 8||Gal4-EED||g034||1||5||2||23.213||1.16065||6.03||9.72
|-
| 9||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
:8 samples, 2 tubes/sample, 1 tube for no template control, total of 17 tubes
 
:{| {{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;"|'''17.7'''
|-
| primer||3||10.5||185.85
|-
| SYBR||7.5||26.25||464.625
|-
| Total||10.5||36.75||650.475
|}
''Step 3: mix gDNA and primer mixes''
:Add 36.75ul primer mix to each sample tube
''Step 4: pipette plate''
:mix tubes immediately before pipetting, add 15 ul from each tube into 3 wells
 
:{| {{table}}
| ||1-3||4-6||7-9||10-12
|-
| A||1 G||1 C||blank-G||
|-
| B||2 G||2 C||blank-C||
|-
| C||3 G||3 C||||
|-
| D||4 G||4 C||||
|-
| E||5 G||5 C||||
|-
| F||6 G||6 C||||
|-
| G||7 G||7 C||||
|-
| H||8 G||8 C||||
|}
 




Revision as of 11:40, 22 May 2015

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05/22/2015

SYBR qPCR to determine plasmid counts for dilution experiment

pX330 primer Primer G
internal primer Primer C
Dilution 2ng/rxn

Step 1: make two PCR tubes for each sample with the following volumes of water and gDNA for 2ng of gDNA per well

' 1rxn x3.5 = 7ng
gDNA x
water to 4.5
Total 4.5 15.75
Tube label Cell type gRNA ug DNA ul lipo rep ng/µL dilute 1:10 ul dilution ul water
1 Gal4-EED g034 0.5 3 1 81.058 4.0529 1.73 14.02
2 Gal4-EED g034 0.5 3 2 86.697 4.33485 1.61 14.14
3 Gal4-EED g034 0.5 5 1 23.89 1.1945 5.86 9.89
4 Gal4-EED g034 0.5 5 2 24.767 1.23835 5.65 10.10
5 Gal4-EED g034 1 3 1 85.32 4.266 1.64 14.11
6 Gal4-EED g034 1 3 2 70.244 3.5122 1.99 13.76
7 Gal4-EED g034 1 5 1 13.387 0.66935 10.46 5.29
8 Gal4-EED g034 1 5 2 23.213 1.16065 6.03 9.72
9 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

8 samples, 2 tubes/sample, 1 tube for no template control, total of 17 tubes
' 1rxn 3.5/triplicate tube 17.7
primer 3 10.5 185.85
SYBR 7.5 26.25 464.625
Total 10.5 36.75 650.475

Step 3: mix gDNA and primer mixes

Add 36.75ul primer mix to each sample tube

Step 4: pipette plate

mix tubes immediately before pipetting, add 15 ul from each tube into 3 wells
1-3 4-6 7-9 10-12
A 1 G 1 C blank-G
B 2 G 2 C blank-C
C 3 G 3 C
D 4 G 4 C
E 5 G 5 C
F 6 G 6 C
G 7 G 7 C
H 8 G 8 C




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