AlanFung:LabNotes/CTCF/2011-6-17
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Objective[edit]
- 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[edit]
TA Cloning[edit]
Ligation of PCR vector[edit]
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[edit]
- Thaw one 50ul vial of One Shot® Competent Cells on ice.
- 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.
- Incubate on ice for 30 minutes. Note: Longer incubations on ice do not seem to have any affect on transformation efficiency.
- Heat-shock the cells for 30 seconds at 42 °C without shaking.
- Immediately transfer the tubes to ice for 2 minutes.
- Add 250 μl of room temperature S.O.C. medium.
- Cap the tube tightly and shake the tube horizontally (256 rpm) at 37°C for 1 hour.
- 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[edit]
DNA pick up 1 colony per well (total of 8 well) 2x Taq MM 25ul M13-21 F+R primer (2.5uM) 4ul H2O 19ul 94C 10 min to break up the cells 94C 30 sec -> (94C for 30 sec, 60C for 30 sec, 72C for 1 min) x30 -> 72C 5 min ->15C forever
- AMpure bead purification and elute in 30ul H2O
- randomly picked up 4 and 8 to be used for sanger sequencing
- PAGE validation (lane 1-8 indicates 8 different colonies)
File:ZhangLab 2 2011-06-16 16hr 31min.jpg
- Picked clone #1-6 for sanger sequencing
- Genewiz sample prep: 3ul DNA + 7ul H2O + 5ul 5uM Forward M13-21 primer
Sequencing Results[edit]
- #5 failed to pass the QC due to non specific results
Blast on Genemapster[edit]
- ssh to genemapster 132.239.135.61
- Create new text file to store new sequence
- vim folder_name
- i to insert
- Copy and paste sequence (separate sequences with >)
- Hit Esc>:wq to save and quit
- To Run Blast
/usr/local/blast-2.2.23/bin/blastall -p blastn -d /media/disk-3/HsGenome/hg18/HsGenome18.bis -i temp/61611clone1_5 -o 61611clone1_5.out
- less file_name to view the results
Results[edit]
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