AlanFung:LabNotes/Sequencing/2009-11-11: Difference between revisions
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==Work on Set 8 DF, 8 FS, 9 DF, 9 FS, 8 PGP1-iPS== | ==Work on Set 8 DF, 8 FS, 9 DF, 9 FS, 8 PGP1-iPS== | ||
==Size Selection== | |||
*Fill any unused well with 30ul EB Buffer | *Fill any unused well with 30ul EB Buffer | ||
*Mix 15ul EB buffer with 0.5ul ladder in a 0.2ml tube | *Mix 15ul EB buffer with 0.5ul ladder in a 0.2ml tube | ||
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[[Image:ZhangLab_2 2009-11-11 14hr 30min.jpg]] | [[Image:ZhangLab_2 2009-11-11 14hr 30min.jpg]] | ||
*Concentrate the DNA with a SpeedVac (no heat) to ~36ul (takes about 1hour). | *Concentrate the DNA with a SpeedVac (no heat) to ~36ul (takes about 1hour). | ||
===Ligation. Blunt-end and TA ligations use the same protocol but slightly different adaptor sequences.=== | |||
* Set up ligation reaction. Note that the ATP in the Quick Ligase buffer hydrolyzes very quickly after several rounds of freeze/thaw cycles, so it’s a good idea to make small aliquots of a fresh tube of Quick Ligase Buffer, and use one small aliquot each time. | |||
{| {{table}} | |||
| ||ul | |||
|- | |||
| End-repaired & size selected DNA||36 | |||
|- | |||
| 40uM adaptor2||2 | |||
|- | |||
| 5X Quick Ligase Buffer||10 | |||
|- | |||
| Quick Ligase||2 | |||
|- | |||
| | |||
|} | |||
*Incubate at room temperature for 15 minutes, purified with MinElute columns, eluted with 20ul EB. |
Revision as of 18:45, 12 November 2009
Rescue CVF-N
- 20ul of CVF-N library, with buffer added instead of iProof master mix
Ethanol Precipitation
- Mix 50ul 100% ethanol, 2ul 3M NaoAC, and 1ul glycoblue
- Store at -80C for 20min
- Centrifuge 10,000 for 20min at 4C
- Discard supernatant
- Add in 750ul 75%ethanol
- Spin 10,000 rpm for 5 min at 4C
- Discard supernatant
- let dry in hood for 10 min
- Add in 21ul ddh2o
Repeat PCR of sequencing library with the following sets
Work on Set 8 DF, 8 FS, 9 DF, 9 FS, 8 PGP1-iPS
Size Selection
- Fill any unused well with 30ul EB Buffer
- Mix 15ul EB buffer with 0.5ul ladder in a 0.2ml tube
- Load 20u end-polished DNA sample into two lanes(use 3 or more lanes if the starting DNA is more than 1ug)
- Run the SizeSelect program for 12~14 min. Pause when the 125bp band just move into the middle collection well
- Use pipette to extract all solution in each of the sample well, and quickly refill the wells with 20ul EB.
- Resume the electrophoresis for additional 15sec, extract DNA and refill the wells with 20ul EB. *Resume the electrophoresis for additional 15sec, extract DNA (all the extracted DNA from the same sample are mixed in a tube)
- Refill the wells with 20ul EB, and run the electrophoresis for additional 3 minutes. Take a picture of the gel to document the extracted fragment sizes.
File:ZhangLab 2 2009-11-11 14hr 28min.jpg File:ZhangLab 2 2009-11-11 14hr 30min.jpg
- Concentrate the DNA with a SpeedVac (no heat) to ~36ul (takes about 1hour).
Ligation. Blunt-end and TA ligations use the same protocol but slightly different adaptor sequences.
- Set up ligation reaction. Note that the ATP in the Quick Ligase buffer hydrolyzes very quickly after several rounds of freeze/thaw cycles, so it’s a good idea to make small aliquots of a fresh tube of Quick Ligase Buffer, and use one small aliquot each time.
ul | |
End-repaired & size selected DNA | 36 |
40uM adaptor2 | 2 |
5X Quick Ligase Buffer | 10 |
Quick Ligase | 2 |
- Incubate at room temperature for 15 minutes, purified with MinElute columns, eluted with 20ul EB.