Alice:LabNotes/2011-4-4
Jump to navigation
Jump to search
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.