AlanFung:LabNotes/CTCF/2011-6-17

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Revision as of 00:22, 18 June 2011 by >Alan6017518 (→‎Ligation of PCR vector)
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Objective

  • Perform Sanger Sequencing on library prepared by Methyl-Seq DNA Sample Prep Kit Sample D 2011-5-16 [[1]]
  • Ensure the library is well constructed and that we are able to generate what Epicenter claimed to be the problem

Protocol

TA Cloning

Ligation of PCR vector

pCR-blunt vector: 1ul

blunt-end DNA:       2ul
2x ligation buffer:  5ul
H2O:                 1ul
Quick T4 DNA Ligase: 1ul
-------------------------
total               10ul

RT for 30min

Transformation

  1. Thaw one 50ul vial of One Shot® Competent Cells on ice.
  2. Add 2 μl ligation product into one vial of One Shot® Chemically Competent E. coli and mix by stirring gently with the pipette tip. Store the rest of the ligation products in -20 C. Do not mix by pipetting up and down.
  3. Incubate on ice for 30 minutes. Note: Longer incubations on ice do not seem to have any affect on transformation efficiency.
  4. Heat-shock the cells for 30 seconds at 42 °C without shaking.
  5. Immediately transfer the tubes to ice for 2 minutes.
  6. Add 250 μl of room temperature S.O.C. medium.
  7. Cap the tube tightly and shake the tube horizontally (256 rpm) at 37°C for 1 hour.
  8. Spread 20 μl from each transformation on a prewarmed selective plate and incubate overnight at 37°C. (To ensure even spreading of small volumes, add 20 μl of S.O.C. medium. The manual recommend that you plate two different volumes to ensure that at least one plate will have well-spaced colonies.)

PCR amplification and preparation for Sanger Sequencing

File:ZhangLab 2 2011-06-16 16hr 31min.jpg

  • Picked clone #1-6 for sanger sequencing

Sequencing Results

Media:06172011_ab1.zip

  • #5 failed to pass the QC due to non specific results

Results

Analysis Clone #1-6 with blast

  • Extract Sequence by 4peaks (Hit the info button>sequence)
  • All clones starts with bunch of Ns and this sequence (partial sequence of pCR 2.1)
gggcgattgggccctctagatgcatgctcgagcggccgccagtgtgatggatatctgcagaattcggct
  • Clones 1, 2, and 3 contains
 CTGTCTCTTATACACATCTCTGA Adapter 2
  • Clone 4 contain
 CTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG Adapter1