AlanFung:LabNotes/CTCF/2010-3-24: Difference between revisions
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>Alan6017518 (Created page with '==Quantification of Jurkat PCR Product== Nanodrop: Qubit: Gel Quant: File:ZhangLab_2 2010-03-24 11hr 35min.jpg') |
>Alan6017518 |
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==Quantification of Jurkat PCR Product== | ==Quantification of Jurkat PCR Product== | ||
Nanodrop: | Nanodrop: 250.7ng/ul | ||
Qubit: | Qubit: 48.4ng/ul | ||
Gel Quant: | Gel Quant: 18.95ng/ul | ||
[[File:ZhangLab_2 2010-03-24 11hr 35min.jpg]] | [[File:ZhangLab_2 2010-03-24 11hr 35min.jpg]] | ||
* I think both the nanaodrop and gel quantification are biased, for the nanaodrop it gives out an extremely high amount of concentration, this usually happens because nanaodrop cannot differentiate between any contamination. As for the gel image the band at 100bp is much stronger than the ladder band which hits that the concentration is higher than 18.95ng/ul. In this case I would go with the qubit quantification. | |||
==Library Prep with new Jurkat PCR product== | |||
*Concentration:48.4ng/ul | |||
*BP:100BP | |||
*setup: 100ng,50ng, 25ng, 10ng, 5ng and -ve control | |||
==End Repair== | |||
*Dilute 10x for 25,10 and 5ng | |||
{| {{table}} | |||
| ||100ng||50ng||25ng||10ng||5ng||-ve | |||
|- | |||
| DNA||2.07||1.03||5.17||2.07||1.03||0 | |||
|- | |||
| End Repair Buffer||10||10||10||10||10||10 | |||
|- | |||
| End Repair Enzyme mix||5||5||5||5||5||5 | |||
|- | |||
| ddH2o||82.93||83.97||79.83||82.93||83.97||85 | |||
|- | |||
| Total Volume||100||100||100||100||100||100 | |||
|- | |||
| | |||
|} | |||
*Incubate at 20C for 30 minutes | |||
*EtOH purification resolve with 37ul ddh2o (250ul 100%EtOH, 10ul 3M NaOH, 1ul Glycoblue) | |||
==A-Tailing== | |||
{| {{table}} | |||
| ||100ng||50ng||25ng||10ng||5ng||-ve | |||
|- | |||
| End Repaired DNA||42||42||42||42||42||42 | |||
|- | |||
| A-tailing buffer||5||5||5||5||5||5 | |||
|- | |||
| Klenow Fragment||3||3||3||3||3||3 | |||
|- | |||
| Total Volume||50||50||50||50||50||50 | |||
|- | |||
| | |||
|} | |||
*Incubate at 37C for 30mins | |||
*EtOH purification resolve with 25ul ddh2o | |||
==Adapter Ligation== | |||
{| {{table}} | |||
| ||100ng||50ng||25ng||10ng||5ng||-ve | |||
|- | |||
| A-tailed DNA||25||25||25||25||25||25 | |||
|- | |||
| Ligation Buffer||10||10||10||10||10||10 | |||
|- | |||
| 1/10x methylation adapter||10||10||10||10||10||10 | |||
|- | |||
| DNA Ligase||5||5||5||5||5||5 | |||
|- | |||
| ddHh2o||50||50||50||50||50||50 | |||
|- | |||
| | |||
|} | |||
*Incubate at 20C for 20mins | |||
*EtOH purification resolve with 30ul ddh2o | |||
==Gel Quantification== | |||
[[File:ZhangLab_2 2010-03-24 21hr 30min.jpg]] | |||
[[File:ZhangLab_2 2010-03-24 21hr 30min-2.jpg]] |
Latest revision as of 04:34, 25 March 2010
Quantification of Jurkat PCR Product[edit]
Nanodrop: 250.7ng/ul Qubit: 48.4ng/ul Gel Quant: 18.95ng/ul
File:ZhangLab 2 2010-03-24 11hr 35min.jpg
- I think both the nanaodrop and gel quantification are biased, for the nanaodrop it gives out an extremely high amount of concentration, this usually happens because nanaodrop cannot differentiate between any contamination. As for the gel image the band at 100bp is much stronger than the ladder band which hits that the concentration is higher than 18.95ng/ul. In this case I would go with the qubit quantification.
Library Prep with new Jurkat PCR product[edit]
- Concentration:48.4ng/ul
- BP:100BP
- setup: 100ng,50ng, 25ng, 10ng, 5ng and -ve control
End Repair[edit]
- Dilute 10x for 25,10 and 5ng
100ng | 50ng | 25ng | 10ng | 5ng | -ve | |
DNA | 2.07 | 1.03 | 5.17 | 2.07 | 1.03 | 0 |
End Repair Buffer | 10 | 10 | 10 | 10 | 10 | 10 |
End Repair Enzyme mix | 5 | 5 | 5 | 5 | 5 | 5 |
ddH2o | 82.93 | 83.97 | 79.83 | 82.93 | 83.97 | 85 |
Total Volume | 100 | 100 | 100 | 100 | 100 | 100 |
- Incubate at 20C for 30 minutes
- EtOH purification resolve with 37ul ddh2o (250ul 100%EtOH, 10ul 3M NaOH, 1ul Glycoblue)
A-Tailing[edit]
100ng | 50ng | 25ng | 10ng | 5ng | -ve | |
End Repaired DNA | 42 | 42 | 42 | 42 | 42 | 42 |
A-tailing buffer | 5 | 5 | 5 | 5 | 5 | 5 |
Klenow Fragment | 3 | 3 | 3 | 3 | 3 | 3 |
Total Volume | 50 | 50 | 50 | 50 | 50 | 50 |
- Incubate at 37C for 30mins
- EtOH purification resolve with 25ul ddh2o
Adapter Ligation[edit]
100ng | 50ng | 25ng | 10ng | 5ng | -ve | |
A-tailed DNA | 25 | 25 | 25 | 25 | 25 | 25 |
Ligation Buffer | 10 | 10 | 10 | 10 | 10 | 10 |
1/10x methylation adapter | 10 | 10 | 10 | 10 | 10 | 10 |
DNA Ligase | 5 | 5 | 5 | 5 | 5 | 5 |
ddHh2o | 50 | 50 | 50 | 50 | 50 | 50 |
- Incubate at 20C for 20mins
- EtOH purification resolve with 30ul ddh2o
Gel Quantification[edit]
File:ZhangLab 2 2010-03-24 21hr 30min.jpg File:ZhangLab 2 2010-03-24 21hr 30min-2.jpg