Haynes Lab:Notebook/CRISPR Editing/2014/07/19

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==07//2014==
==07//2014==
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'''QPCR of genomic DNA isolated from KAH154 U20S cells'''
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<br><br>
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{| {{table}}
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| align="center" style="background:#f0f0f0;"|''''''
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| align="center" style="background:#f0f0f0;"|'''Template DNA'''
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| align="center" style="background:#f0f0f0;"|'''Gene Target (primers)'''
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| align="center" style="background:#f0f0f0;"|'''Technical Replicates'''
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|-
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| Rxn 1||KAH154 U20S genomic DNA||Target 1 - primers flanking stop codon insertion site at 72bp||3
 +
|-
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| Rxn 2||KAH154 U20S genomic DNA||Target 2 - primers flanking stop codon insertion site at 162bp||3
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|-
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| Rxn 3||KAH154 U20S genomic DNA||Target 3 - primers flanking stop codon insertion site at 255bp||3
 +
|-
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| Rxn 4||KAH154 U20S genomic DNA||GAPDH - primers from KAH||3
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|-
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| Rxn 5||no template||Target 1 - primers flanking stop codon insertion site at 72bp||3
 +
|-
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| Rxn 6||no template||Target 2 - primers flanking stop codon insertion site at 162bp||3
 +
|-
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| Rxn 7||no template||Target 3 - primers flanking stop codon insertion site at 255bp||3
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|-
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| Rxn 8||no template||GAPDH - primers from KAH||3
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|}
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<br><br>
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''Total wells needed = 8 x 3 = 24; This experiment can be done in one 96-well plate.''
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<br>
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[[Image: 14.07.19 qPCR plate layout.png| 500px | plate layout]]
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 +
'''Gene Target (primer) master mixes'''
 +
 +
Gene Targets: 4 tubes
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#Target 1 - primers flanking stop codon insertion site at 72bp
 +
#Target 2 - primers flanking stop codon insertion site at 162bp
 +
#Target 3 - primers flanking stop codon insertion site at 255bp
 +
#GAPDH - primers from KAH
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<br>
 +
Gene Target Master Mix
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{| {{table}}
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| align="center" style="background:#f0f0f0;"|'''Reagent'''
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| align="center" style="background:#f0f0f0;"|'''(Single well)'''
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| align="center" style="background:#f0f0f0;"|'''x6.5'''
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|-
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| 2x SYBR Green Master Mix||(7.5 µL)||48.75
 +
|-
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| 750 nM F/R primer mix||(3.0 µL)||19.5
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|-
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| Total vol.||(10.5 µL)||68.25
 +
|}
 +
<br>
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'''Genomic DNA Templates'''
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<br>
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Templates: 2 tubes
 +
#untreated KAH154 U20S cells
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#no template
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<br>
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Template Master Mix
 +
{| {{table}}
 +
| align="center" style="background:#f0f0f0;"|'''Reagent'''
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| align="center" style="background:#f0f0f0;"|'''(Single well)'''
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| align="center" style="background:#f0f0f0;"|'''x12.5'''
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|-
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| DNA or PCR H2O*||(0.067 µL)||0.8375
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|-
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| PCR H2O||(4.433 µL)||55.4125
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|-
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| Total vol.||(4.5 µL)||56.25
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|}
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<br><br>
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'''Loading the 96-well plate'''
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#Pipette 13.5 µL of the appropriate Template master mix into the first well of each 3-well group.
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#Add 31.5 µL of the appropriate Gene Target master mix each Template mix in the first well of each 3-well group. After each addition, mix by gently pipetting up and down 3 - 5 times without making bubbles.
 +
#Transfer 15 µL of solution from the first well in each 3-well group to the second and third wells (e.g., A1 into A2, and A3). Using a fresh pipette tip for each 3-well group, to do the same for A4-6, A7-8, etc.
 +
#Seal the plate with clear film.
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<br><br>
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Ran on the Haynes lab qPCR machine with melt curve<br><br>
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'''Results'''
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<br>
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No template wells did not show any significant fluorescence or peaks on melt curve <br>
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KAH154 U20S genomic DNA
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''finish table''
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{| {{table}}
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| align="center" style="background:#f0f0f0;"|'''Primer set'''
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| align="center" style="background:#f0f0f0;"|'''Cp'''
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| align="center" style="background:#f0f0f0;"|'''Melt  curve Tm'''
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| align="center" style="background:#f0f0f0;"|'''Melt curve area'''
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|-
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| Target 1 - primers flanking stop codon insertion site at 72bp||||||
 +
|-
 +
| Target 2 - primers flanking stop codon insertion site at 162bp||||||
 +
|-
 +
| Target 3 - primers flanking stop codon insertion site at 255bp||||||
 +
|-
 +
| GAPDH - primers from KAH||||||
 +
|}
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<br>
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'''Conclusions'''
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#Order new primers for Target 1
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#Targets 2 and 3 work well
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#GAPDH had a really late Cp and a lower melting temperature. Will test some from KAH's set here: http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/Polycomb_project/2010/10/17
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<br><br>
-
[[Image: PCR layout.png‎ | 500px | plate layout]]
 
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

Revision as of 19:24, 20 July 2014

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07//2014

QPCR of genomic DNA isolated from KAH154 U20S cells

' Template DNA Gene Target (primers) Technical Replicates
Rxn 1KAH154 U20S genomic DNATarget 1 - primers flanking stop codon insertion site at 72bp3
Rxn 2KAH154 U20S genomic DNATarget 2 - primers flanking stop codon insertion site at 162bp3
Rxn 3KAH154 U20S genomic DNATarget 3 - primers flanking stop codon insertion site at 255bp3
Rxn 4KAH154 U20S genomic DNAGAPDH - primers from KAH3
Rxn 5no templateTarget 1 - primers flanking stop codon insertion site at 72bp3
Rxn 6no templateTarget 2 - primers flanking stop codon insertion site at 162bp3
Rxn 7no templateTarget 3 - primers flanking stop codon insertion site at 255bp3
Rxn 8no templateGAPDH - primers from KAH3



Total wells needed = 8 x 3 = 24; This experiment can be done in one 96-well plate.
plate layout

Gene Target (primer) master mixes

Gene Targets: 4 tubes

  1. Target 1 - primers flanking stop codon insertion site at 72bp
  2. Target 2 - primers flanking stop codon insertion site at 162bp
  3. Target 3 - primers flanking stop codon insertion site at 255bp
  4. GAPDH - primers from KAH


Gene Target Master Mix

Reagent (Single well) x6.5
2x SYBR Green Master Mix(7.5 µL)48.75
750 nM F/R primer mix(3.0 µL)19.5
Total vol.(10.5 µL)68.25


Genomic DNA Templates
Templates: 2 tubes

  1. untreated KAH154 U20S cells
  2. no template


Template Master Mix

Reagent (Single well) x12.5
DNA or PCR H2O*(0.067 µL)0.8375
PCR H2O(4.433 µL)55.4125
Total vol.(4.5 µL)56.25



Loading the 96-well plate

  1. Pipette 13.5 µL of the appropriate Template master mix into the first well of each 3-well group.
  2. Add 31.5 µL of the appropriate Gene Target master mix each Template mix in the first well of each 3-well group. After each addition, mix by gently pipetting up and down 3 - 5 times without making bubbles.
  3. Transfer 15 µL of solution from the first well in each 3-well group to the second and third wells (e.g., A1 into A2, and A3). Using a fresh pipette tip for each 3-well group, to do the same for A4-6, A7-8, etc.
  4. Seal the plate with clear film.



Ran on the Haynes lab qPCR machine with melt curve

Results
No template wells did not show any significant fluorescence or peaks on melt curve
KAH154 U20S genomic DNA finish table

Primer set Cp Melt curve Tm Melt curve area
Target 1 - primers flanking stop codon insertion site at 72bp
Target 2 - primers flanking stop codon insertion site at 162bp
Target 3 - primers flanking stop codon insertion site at 255bp
GAPDH - primers from KAH


Conclusions

  1. Order new primers for Target 1
  2. Targets 2 and 3 work well
  3. GAPDH had a really late Cp and a lower melting temperature. Will test some from KAH's set here: http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/Polycomb_project/2010/10/17





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