Alice:LabNotes/2011-4-4

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Revision as of 22:09, 5 April 2011 by >Zsakura2 (Created page with '=Prepare genomic DNA for hybridization (for use with Nimblegen's kit)= *received a new batch of cell pellet (HGPS only) on 3/31/2011, finished the DNA extraction on 4/1/2011 *she…')
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Prepare genomic DNA for hybridization (for use with Nimblegen's kit)

  • received a new batch of cell pellet (HGPS only) on 3/31/2011, finished the DNA extraction on 4/1/2011
  • sheared the 3ug of DNA at UCSD Moores Cancer Center (Frazer Lab)
  • sample to be processed: HGPS

End-repair Reactions

Fragmented DNA                             85 ul
10X End Repair Reaction Buffer             11 ul
End Repair Enzyme Mix                      4  ul
Keep the tube at room temperature (~20°C) for 30 minutes.
Purify with Qiaquick column and elute in 40ul ddH2O
Note: for blunt-end ligation, the A-Tailing reaction should be skipped. Doing TA ligation is preferable since it eliminates the
chance of getting chimeric reads.
Nanodrop result (40ul total for each):
HGPS: 45ng/ul

A-Tailing reactions

Blunt-end DNA                             40 ul
10X dA-Tailing Reaction Buffer (10X)       5 ul
Klenow Fragment (3’-5’ exo-)               3 ul
H2O                                        2 ul
Incubated at 37C for 30min
purified the products with Qiaquick column and elute in 30ul ddH2O
Nanodrop result (30ul total for each):
HGPS: 30ng/ul

adaptor ligation

Prepare adaptors (need to be done only for the first time): 
100uM PE-t:          20ul
100uM PE-b:          20ul
10x stoffel buffer:  10ul
H2O:                 50ul
94C for 3 min, and then cool down to 20C at the rate of 0.1C/sec. 
commonly used adaptors:
Blunt-end adaptors:
5’NH2- ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’OH         Solexa_1_up
3’NH2-ATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA-5’OH            Solexa_1_lo_nop

TA adaptors (for the one adaptor protocol):
5- CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT-3’OH        PE_t_adapter
3-CTTCTGCCGTATGCTCGAGAAGGCTAG-5’Phos                    t_adaptor_rc_s
  
regular Y adaptor:
PE_t_adaptor(top)              ACACTCTTTCCCTACACGACGCTCTTCCGATC*T              3'-Phosphorothioate bond    
PE_b_adaptor(bottom)           \\5phos\\GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG       5'-phosphorylation    
  • 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. target:adaptor molar ratio is 1:15
A-tailed DNA:        30ul
5x ligation buffer:  12ul
20uM Y adaptor:      11ul
Quick T4 Ligase:      5ul
Incubate at room temperature for 30 minutes
purify the product with Qiaquick columnes and elute in 40ul ddH2O

PCR amplification

10ul of ligated DNA for each well, and a total of 4 well
Ligation products                 10ul  
100uM PCR_F                       0.2ul        
100uM PCR_R                       0.2ul
2X phusion HF master mix          50ul     
H2O                               40ul       
PCR program: 98 °C 30sec  -> 7 cycles of (98°C 10sec -> 60°C 20 sec -> 72°C 15sec) -> 72°C 3min ->15°C hold. 
purify the products with Qiaquick columns and elute in 40ul ddH2O
  • Nanodrop result:
  • HGPS: 34ng/ul * 40ul

Results

  • The yield of the last PCR step is very low compare to the previous whole-genome libraries that I have prepared.