AlanFung:LabNotes/Sequencing/2009-11-11: Difference between revisions

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>Alan6017518
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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==
*Start from Size Selection
==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.