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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: 46.6ng/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: 69.4ng/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-2: 69.2ng/ul * 40ul *[[File:ZhangLab_2 2011-04-05 18hr 05min.jpg]] ===Results=== *The yield of the last PCR step is very low compare to the previous whole-genome libraries that I have prepared. However, the result agrees with the whole-genome library of the same sample I prepared on 3/28/2011. Although the results aren't as good as the previous libraries that I have prepared, but we should still be able to get useful information after sequencing. Therefore I will combine the library from 3/28/2011 with this batch to do liquid capture. ===Hybrid Capture using Nimblegen kit (4/6/2011)=== *3/28/2011 batch: HGPS- 34ng/ul * 60ul *4/4/2011 batch: HGPS- 69.2ng/ul * 40ul # Add 5μl of 1mg/ml COT DNA and 2.5μg of amplified sample library to a new 1.5ml tube. # Add 10μl of each 100μM PE-HE1 and PE-HE2 Oligos to the amplified sample library plus COT DNA. (PE-HE1=PCR_F, PE-HE2=PCR_R) # Close the tube’s lid and make a hole in the top of the tube’s cap with an 18 - 20 gauge or smaller needle. (Note: The hole in the top of the tube’s cap is a precaution to suppress contamination in the SpeedVac.) # Dry the amplified sample library/COT DNA/PE-HE Oligos in a SpeedVac on high heat (60°C). (Note: This step may take 30 minutes or longer. To minimize drying time, dry COT DNA ahead of time and then add the amplified sample library and PE- HE Oligos. Denaturation of the DNA with high heat is not problematic after linker ligation because the hybridization utilizes single-stranded DNA.) # To each dried-down amplified sample library/COT DNA/PE-HE Oligos, add 7.5μl of 2X SC Hybridization Buffer and 3μl of SC Hybridization Component A # Cover the hole in the tube’s cap with a sticker or small piece of laboratory tape. Vortex the amplified sample library/COT DNA/PE-HE Oligos plus Hybridization Cocktail (2X SC Hybridization Buffer + SC Hybridization Component A) for 10 seconds and centrifuge at maximum speed for 10 seconds. # Place the amplified sample library/COT DNA/PE-HE Oligos/Hybridization Cocktail in a 95°C heat block for 10 minutes to denature the DNA. # Centrifuge the amplified sample library/COT DNA/PE-HE/Hybridization Cocktail at maximum speed for 10 seconds at room temperature. # Transfer the amplified sample library/COT DNA/PE-HE Oligos/Hybridization Cocktail to the 4.5μl aliquot of Exome Library in a 0.2ml PCR tube prepared in Chapter 2 (entire volume can also be transferred to a 96-well PCR plate). Vortex for 3 seconds and centrifuge at maximum speed for 10 seconds. # Incubate in a thermocycler (with heated lid turned on at 57C) at 47°C for 72 hours.
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