AlanFung:LabNotes/CTCF/2010-1-15: Difference between revisions
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Prepare 7 Qubit Assay tubes | Prepare 7 Qubit Assay tubes | ||
Prepare 7*200ul diluted dye=1400ul | Prepare 7*200ul diluted dye=1400ul | ||
Mix 7ul dye with 1393ul buffer | Mix 7ul dye with 1393ul buffer | ||
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.