Alice:LabNotes/2010-8-5
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Prepare genomic DNA for hybridization (for use with Nimblegen's kit)
- the following samples will be processed:
- KiPS
- Human HUVEC, HUViPS 4F-1, HUViPS 4F-3
- FiPS
- Note: HUV ips 4F-3 came back with only 70ul, therefore i added 15ul of ddH2O to it. The rest of the samples were around 85-90 ul
End-repair Reactions
Fragmented DNA 85 ul 10X End Repair Reaction Buffer 10 ul End Repair Enzyme Mix 5 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):
A-Tailing reactions (done on 5/22/2010)
Blunt-end DNA 40 ul 10X dA-Tailing Reaction Buffer (10X) 5 ul Klenow Fragment (3’-5’ exo-) 2 ul H2O 3 ul Incubated at 37C for 30min purified the products with Qiaquick column and elute in 40ul ddH2O
- Nanodrop result (40ul total for each):
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. adaptor:target molar ratio is 1:10~20 For HVEC, HUV ips 4F1, HUV ips 4F3: A-tailed DNA 40 ul 20uM Y adaptor 2 ul 5X Quick ligase buffer 10 ul Quick Ligase 2 ul
For HFFxF, Fips 3F#1: A-tailed DNA 20 ul 20uM Y adaptor 3 ul 5X Quick ligase buffer 10 ul Quick Ligase 2 ul H2O 15 ul
For KERAT, Kips 4F#8: A-tailed DNA 20 ul 20uM Y adaptor 5 ul 5X Quick ligase buffer 10 ul Quick Ligase 2 ul H2O 13 ul
Incubate at room temperature for 20 minutes purify the product with Agencourt AMpure kit and elute in 40ul ddH2O for HVEC, HUV ips 4F1, HUV ips 4F3 (60ul for the rest)
PCR amplification
Ligation products 10ul 100uM PCR_F 0.2ul 100uM PCR_R 0.2ul 2X phusion HF master mix 50ul 50X SYBR Green I 0.4ul H2O 45ul 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 Ampure beads and elute in 160ul for 3 HUVEC lines, and 240ul for the rest
- nanodrop result: