AlanFung:LabNotes/CTCF/2011-6-6: Difference between revisions

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>Alan6017518
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{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''Amount per reaction'''
| align="center" style="background:#f0f0f0;"|'''amount per reaction'''
| align="center" style="background:#f0f0f0;"|'''x6.6'''
|-
|-
| Distilled water||31||204.6
| distilled water||34
|-
|-
| 10X pfuTurbo Cx reaction buffer||5||33
| 10x pfuturbo cx reaction buffer||5
|-
|-
| dNTPs (10mM each DNTP)||1||6.6
| dNTPs(10mM each DNTP)||1
|-
|-
| DNA template/STD||5||
| DNA template||2
|-
|-
| CHR8 F Primer (3.3uM)||3||
| CHR8 F primer 3.3uM||3
|-
|-
| CHR8 R Primer (3.3uM)||3||
| CHR8 R primer 3.3um||3
|-
|-
| SYBR Green (50X)||1||6.6
| sybr green 50x||1
|-
|-
| PfuTurbo Cx Hotstart DNA Polymerase (2.5U/ul)||1||6.6
| pfuturbo cx hotstart dna polymerase 2.5U/ul||1
|-
|-
| Total reaction volume||50||257.4
| total reaction volume||50
|}
|}
*Add 39ul master mix to primers and templates
*Add 39ul master mix to primers and templates

Revision as of 20:52, 6 June 2011

Perform bisulfite conversion on 200ng of gDNA

Protocol

Setup PCR Reaction

  • Add the components on ice in order while mixing gently.
Component amount per reaction
distilled water 34
10x pfuturbo cx reaction buffer 5
dNTPs(10mM each DNTP) 1
DNA template 2
CHR8 F primer 3.3uM 3
CHR8 R primer 3.3um 3
sybr green 50x 1
pfuturbo cx hotstart dna polymerase 2.5U/ul 1
total reaction volume 50
  • Add 39ul master mix to primers and templates
    • Perform Manufacturer protocol for PfuTurbo Cx Hotstart DNA Polymerase
95C 2m
95C 30s
52C 30s
72C 2m
Plate Read
Go to step 2 repeat for 29 times
72C 10m
15C hold

Results