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

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>Alan6017518
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==Perform bisulfite conversion on 200ng of gDNA==
==Perform bisulfite conversion on 200ng of gDNA==
==Protocol==
===Setup PCR Reaction===
*Add the components on ice in order while mixing gently.
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''Amount per reaction'''
| align="center" style="background:#f0f0f0;"|'''x6.6'''
|-
| Distilled water||31||204.6
|-
| 10X pfuTurbo Cx reaction buffer||5||33
|-
| dNTPs (10mM each DNTP)||1||6.6
|-
| DNA template/STD||5||
|-
| CHR8 F Primer (3.3uM)||3||
|-
| CHR8 R Primer (3.3uM)||3||
|-
| SYBR Green (50X)||1||6.6
|-
| PfuTurbo Cx Hotstart DNA Polymerase (2.5U/ul)||1||6.6
|-
| Total reaction volume||50||257.4
|}
*Add 39ul master mix to primers and templates
**Perform Manufacturer protocol for PfuTurbo Cx Hotstart DNA Polymerase
95C 2m
95C 30s
58C 30s
72C 1m
Plate Read
Go to step 2 repeat for 29 times
72C 10m
15C hold
==Results==

Revision as of 20:37, 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 x6.6
Distilled water 31 204.6
10X pfuTurbo Cx reaction buffer 5 33
dNTPs (10mM each DNTP) 1 6.6
DNA template/STD 5
CHR8 F Primer (3.3uM) 3
CHR8 R Primer (3.3uM) 3
SYBR Green (50X) 1 6.6
PfuTurbo Cx Hotstart DNA Polymerase (2.5U/ul) 1 6.6
Total reaction volume 50 257.4
  • Add 39ul master mix to primers and templates
    • Perform Manufacturer protocol for PfuTurbo Cx Hotstart DNA Polymerase
95C 2m
95C 30s
58C 30s
72C 1m
Plate Read
Go to step 2 repeat for 29 times
72C 10m
15C hold

Results