AlanFung:LabNotes/CTCF/2010-1-15: Difference between revisions

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>Alan6017518
(New page: ==Quantify Bisulfite Converted Samples with Qubit ssDNA Kit== ==On 50ng, 10ng, 5ng, 1ng, 0.5ng, 0.1ng & 0.05ng P21-200BP== 7 samples Prepare 7 Qubit Assay tubes Prepare 7*200ul dilute...)
 
>Alan6017518
 
(11 intermediate revisions by 2 users not shown)
Line 4: Line 4:
  Prepare 7 Qubit Assay tubes
  Prepare 7 Qubit Assay tubes
   
   
  Prepare 7*200ul diluted dye=840ul
 
  Mix 4.2ul dye with 835.8ul buffer
  Prepare 7*200ul diluted dye=1400ul
  Mix 7ul dye with 1393ul buffer


  Mix 2ul samples with 98ul diluted dye
  Mix 2ul samples with 98ul diluted dye
Line 11: Line 12:
   
   
  Result
  Result
{| {{table}}
==PCR quantification of Bisulfite treated samples using 3 paris of CHR primers==
| Start (ng)||Sample Concentration ng/ul||Volume||Amount Recovered (ng)||Yield %
|-
| 50||1.7||20||34||68
|-
| 10||0.25||20||5||50
|-
| 5||0.13||20||2.6||52
|-
| 1||0.26||20||5.2||520
|-
| 0.5||N/A||20||||
|-
| 0.1||N/A||20||||
|-
| 0.05||N/A||20||||
|-
|
|}
 
==PCR quantification of Bisulfite treated samples using 3 paris of CHR bilsulfite conversion primers==
 
==On 50ng, 10ng, 5ng, 1ng, 0.5ng, 0.1ng & 0.05ng P21-200BP==
==On 50ng, 10ng, 5ng, 1ng, 0.5ng, 0.1ng & 0.05ng P21-200BP==
{| {{table}}
| 1 sample||CHR22||CHR21||CHR8
|-
| 2X iQ Super Mix||20||20||20
|-
| Primer F (3.3uM)||6||6||6
|-
| Primer R (3.3uM)||6||6||6
|-
| ||||||
|-
| 7 Samples||CHR22||CHR21||CHR8
|-
| 2X iQ Super Mix||160||160||160
|-
| Primer F (3.3uM)||48||48||48
|-
| Primer R (3.3uM)||48||48||48
|-
| ||||||
|-
| ||Setup||||
|-
| ||CHR22||CHR21||CHR8
|-
| 1||50ng||50ng||50ng
|-
| 2||10ng||10ng||10ng
|-
| 3||5ng||5ng||5ng
|-
| 4||1ng||1ng||1ng
|-
| 5||0.5ng||0.5ng||0.5ng
|-
| 6||0.1ng||0.1ng||0.1ng
|-
| 7||0.05ng||0.05ng||0.05ng
|-
|
|}
*Load 2ul sample with 32ul mix
Perform PCR reaction in thermocycler
 
      Step1  96C, 3m
      Step2  95C, 30s
      Step3  62C, 1m
      Step4  72C, 1m
      Step5  Go to step2 repeat 39 times
      Step6  72C, 5m
      Step7  4C,  Forever
*Only 50ng,10ng, 5ng was amplified based on the QPCR curve, will run a gel to quantify
==Run Size Select gel to quantify==
*Load 20ul samples into well
*Mix 15ul EB buffer with 0.5ul low mass ladder in a 0.2ml tube
{| {{table}}
| Setup||||||||||||||
|-
| 1||2||3||4||5||6||7||8
|-
| 50ng||||||10ng||||||5ng||
|-
| CHR22||CHR21||CHR8||CHR22||CHR21||CHR8||CHR22||CHR21
|-
| ||||||||||||||
|-
| 9||10||11||12||13||14||15||16
|-
| 5ng||1ng||||||0.5ng||||||0.1
|-
| CHR8||CHR22||CHR21||CHR8||CHR22||CHR21||CHR8||CHR21
|-
|
|}
[[File:ZhangLab_2 2010-01-19 15hr 14min.jpg]]
*Based on the gel image the bisulfite conversion seems to be incomplete, repeat conversion with no washing step.

Latest revision as of 22:47, 20 January 2010

Quantify Bisulfite Converted Samples with Qubit ssDNA Kit[edit]

On 50ng, 10ng, 5ng, 1ng, 0.5ng, 0.1ng & 0.05ng P21-200BP[edit]

7 samples
Prepare 7 Qubit Assay tubes

Prepare 7*200ul diluted dye=1400ul
Mix 7ul dye with 1393ul buffer
Mix 2ul samples with 98ul diluted dye
vortex spin down and wait for 2 mins

Result
Start (ng) Sample Concentration ng/ul Volume Amount Recovered (ng) Yield %
50 1.7 20 34 68
10 0.25 20 5 50
5 0.13 20 2.6 52
1 0.26 20 5.2 520
0.5 N/A 20
0.1 N/A 20
0.05 N/A 20

PCR quantification of Bisulfite treated samples using 3 paris of CHR bilsulfite conversion primers[edit]

On 50ng, 10ng, 5ng, 1ng, 0.5ng, 0.1ng & 0.05ng P21-200BP[edit]

1 sample CHR22 CHR21 CHR8
2X iQ Super Mix 20 20 20
Primer F (3.3uM) 6 6 6
Primer R (3.3uM) 6 6 6
7 Samples CHR22 CHR21 CHR8
2X iQ Super Mix 160 160 160
Primer F (3.3uM) 48 48 48
Primer R (3.3uM) 48 48 48
Setup
CHR22 CHR21 CHR8
1 50ng 50ng 50ng
2 10ng 10ng 10ng
3 5ng 5ng 5ng
4 1ng 1ng 1ng
5 0.5ng 0.5ng 0.5ng
6 0.1ng 0.1ng 0.1ng
7 0.05ng 0.05ng 0.05ng
  • Load 2ul sample with 32ul mix

Perform PCR reaction in thermocycler

      Step1   96C, 3m
      Step2   95C, 30s
      Step3   62C, 1m
      Step4   72C, 1m
      Step5   Go to step2 repeat 39 times
      Step6   72C, 5m
      Step7   4C,  Forever
  • Only 50ng,10ng, 5ng was amplified based on the QPCR curve, will run a gel to quantify

Run Size Select gel to quantify[edit]

  • Load 20ul samples into well
  • Mix 15ul EB buffer with 0.5ul low mass ladder in a 0.2ml tube
Setup
1 2 3 4 5 6 7 8
50ng 10ng 5ng
CHR22 CHR21 CHR8 CHR22 CHR21 CHR8 CHR22 CHR21
9 10 11 12 13 14 15 16
5ng 1ng 0.5ng 0.1
CHR8 CHR22 CHR21 CHR8 CHR22 CHR21 CHR8 CHR21

File:ZhangLab 2 2010-01-19 15hr 14min.jpg

  • Based on the gel image the bisulfite conversion seems to be incomplete, repeat conversion with no washing step.