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==
*Start from Size Selection
==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.
[[Image:ZhangLab_2 2009-11-11 14hr 28min.jpg]]
[[Image:ZhangLab_2 2009-11-11 14hr 28min.jpg]]
[[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).
===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 22ul EB.
==PCR of sequencing library==
*Include CVF-N set
*Total 6 sets of libraries
Solexa_PCR_up: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
AmpR6.3Sol:  CAAGCAGAAGACGGCATACGAGCTCTTCGGAACGATGAGCCTCCAAC
AmpF6.3rSol: CAAGCAGAAGACGGCATACGAGCTCTTCCAGATGTTATCGAGGTCCGA
{| {{table}}
| Ligation products||10||10
|-
| 10uM solexa PCR up||2||2
|-
| 10uM AmpR6.3Sol||2||-
|-
| 10uM AmpF6.3Sol||-||2
|-
| 2X iProof master mix (Bio-Rad)||50||50
|-
| 50X SYBR Green I||0.4||0.4
|-
| H2O||36||36
|-
|
|}
*Setup to master mix
{| {{table}}
| ||F||R
|-
| Ligation products||||
|-
| 10uM solexa PCR up||13.2||13.2
|-
| 10uM AmpR6.3Sol||13.2||-
|-
| 10uM AmpF6.3Sol||-||13.2
|-
| 2X iProof master mix (Bio-Rad)||330||330
|-
| 50X SYBR Green I||2.64||2.64
|-
| H2O||237.6||237.6
|-
|
|}
PCR program: 98 °C 30sec -> 8 cycles of (98°C 10sec -> 60°C 20 sec -> 72°C 15sec) -> 72°C 3min ->15°C hold.
                                                                                                             
Mix the amplicons with two sets of primers, purified with Qiaquick columns. Typically, the PCR products are clean enough for sequencing without further size selection.

Latest revision as of 19:00, 12 November 2009

Rescue CVF-N[edit]

  • 20ul of CVF-N library, with buffer added instead of iProof master mix

Ethanol Precipitation[edit]

  • 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[edit]

Size Selection[edit]

  • 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.[edit]

  • 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 22ul EB.

PCR of sequencing library[edit]

  • Include CVF-N set
  • Total 6 sets of libraries

Solexa_PCR_up: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT

AmpR6.3Sol: CAAGCAGAAGACGGCATACGAGCTCTTCGGAACGATGAGCCTCCAAC

AmpF6.3rSol: CAAGCAGAAGACGGCATACGAGCTCTTCCAGATGTTATCGAGGTCCGA

Ligation products 10 10
10uM solexa PCR up 2 2
10uM AmpR6.3Sol 2 -
10uM AmpF6.3Sol - 2
2X iProof master mix (Bio-Rad) 50 50
50X SYBR Green I 0.4 0.4
H2O 36 36
  • Setup to master mix
F R
Ligation products
10uM solexa PCR up 13.2 13.2
10uM AmpR6.3Sol 13.2 -
10uM AmpF6.3Sol - 13.2
2X iProof master mix (Bio-Rad) 330 330
50X SYBR Green I 2.64 2.64
H2O 237.6 237.6

PCR program: 98 °C 30sec -> 8 cycles of (98°C 10sec -> 60°C 20 sec -> 72°C 15sec) -> 72°C 3min ->15°C hold.


Mix the amplicons with two sets of primers, purified with Qiaquick columns. Typically, the PCR products are clean enough for sequencing without further size selection.