Jie:LabNotes/CpgSeq/2009-6-22
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Methyl_PCR for ASM validation[edit]
PCR on bisuphite conversed Jurkat-DNA[edit]
2x iQ supermix 20 x 12 Primer F (50uM) 0.5 0.5 each Primer R (50uM) 0.5 0.5 each BS Jurkat gDNA (202ng/ul) 0.1 1.2 dH2O 20 240 total 40ul
The primers are 20061015_ASM_valid. I set the annealing temperature at 58C, 62C. 96C 3min-> 40x (95C 30sec, 58C or 62C 1min, 72C 1min)-> 72C 5min.
File:20090622 ASM 58C anneal.jpg20090622_ASM_58C anneal
File:20090622 ASM 62C anneal.jpg20090622_ASM_62C anneal
PCR on samples[edit]
The following two genes demonstrate the ASM in all samples that SNPs were called. LSP1: PGP1_F and IMR90_iPS MATN4: PGP1_L and PGP3_L
The following two genes demonstrate the ASM in specific tissues: DNMT3B: PGP3L and IMR90_iPS MMP9: PGP1_L and PGP1_F
The bisulfite comverted gDNA comes from: IMR09_iPS: 83.5ng/ul refer to LabNotes on [1] PGP1_F: 119.5ng/ul refer to LabNotes on [2] PGP1_L: 386.7ng/ul refer to LabNotes on [3] PGP3_L: 238.6ng/ul refer to LabNotes on [4]
2x iQ supermix 20 x 8 Primer F (50uM) 0.5 0.5 each Primer R (50uM) 0.5 0.5 each BS Jurkat gDNA (202ng/ul) 0.1 0.8 dH2O 20 160 total 40ul
96C 3min-> 37x (95C 30sec, 58C or 62C 1min, 72C 1min)-> 72C 5min. 58C for MATN4 and DNMT3B; 62C for LSP1 and MMP9.
File:20090623 ASM valid.jpg20090623_ASM_valid
TA cloning of the above 8 samples[edit]
Step 1. Cloning into pCR2.1.
Set up the 10ul ligation reaction as follows: x 9 DNA template 0.5ul 4.5 10x Ligation Buffer 0.5ul 4.5 pCR2.1 vector(25ng/ul) 1ul 9 water 2.5ul 22.5 T4 DNA ligase(4 Weiss Units)0.5ul 4.5
Incubate the ligation reaction at 14C overnight.
Step 2.Transforming TOP10 E. coli
1.Equilibrate a water bath to 42C. 2.Bring the SOC medium to room temperature. 3.Take LB plate containing antibotics at 37C for 30 mins. Spread the plate with 80ul of 20mg/ml X_Gal. 4.Centrifuge vials containing the ligation reactions briefly and place them on ice. 5.Thaw on ice one 50ul vial of frozen OneShot TOP10 E coli. 6.Pipette 2ul of each ligation reaction directly into the vial of competent cells and mix by stirring gently with the pipette tip. 7.Incubate the vials on ice for 30 mins. Store the remaining ligation mixtures at -20C. 8.Heat shock the cells for 30 seconds at 42 C without shaking. Immediately transfer the vials to ice. 9.Add 250ul of room temperature SOC medium into each vial. 10.Shake the vials horizontally at 37C for 1 hour at 225rpm in a shaking incubator. 11.Spread 10ul to 50ul from each transformation bial on LB agar plates containing X_Gal and 50ug/ml of kanamycin. Be sure to plate two different volumes to ensure the at least one plate has well spaced colonies. 12.Incubate plate overnight at 37C.
Blunt clone and transformation of captured targets and shotgun sequencing library[edit]
samples: 21070*1 100406_97k captured targets (96nM) refer to LabNotes on [5] No09_9_97k_shotgun_library: refer to LabNotes on [6] No09_9_97k_shotgun_library: 2ng/ul (15nM) refer to LabNotes on [7]
Set up the 10ul ligation reaction as follows: x3 PCR product of sequencing library 1ul 10x Ligation Buffer 0.5ul pCR2.1 vector(25ng/ul) 1ul water 2ul T4 DNA ligase(4 Weiss Units) 0.5ul
Incubate the ligation reaction at 16C 1h.
Transforming TOP10 E. coli 1.Equilibrate a water bath to 42C. 2.Bring the SOC medium to room temperature. 3.Take LB plate containing antibotics at 37C for 30 mins. Spread the plate with 80ul of 20mg/ml X_Gal. 4.Centrifuge vials containing the ligation reactions briefly and place them on ice. 5.Thaw on ice one 50ul vial of frozen OneShot TOP10 E coli. 6.Pipette 2ul of each ligation reaction directly into the vial of competent cells and mix by stirring gently with the pipette tip. 7.Incubate the vials on ice for 30 mins. Store the remaining ligation mixtures at -20C. 8.Heat shock the cells for 45 seconds at 42 C without shaking. Immediately transfer the vials to ice. 9.Add 250ul of room temperature SOC medium into each vial. 10.Shake the vials horizontally at 37C for 1 hour at 225rpm in a shaking incubator. 11.Spread 10ul to 50ul from each transformation bial on LB agar plates containing X_Gal and 50ug/ml of kanamycin. Be sure to plate two different volumes to ensure the at least one plate has well spaced colonies. 12.Incubate plate overnight at 37C.
I sent 3 96-well plates containing the three blunt clones to Agencourt on 09-06-25. I sent one 96-well plate containing the clones from DMNT3B and MMP9 together with the forward and reverse primers to Agencourt on 09-06-25. I sent one 96-well palte containg the clones from LSP1 and MATN4 together with the forward and reverse primers to Agencourt on 09-06-29.
results of clonings from cpg97k captured targets[edit]
Totally I got 93 reads. 75 hits were achieved when I mapped the reads to targets sequences. 35 out of 75 hits were specific capture (46.7%). Among these 35 hits, 5 contain only one capture arms, 2 contain two capture arms from adjacent two probes. There are 2 cloncal reads. Among these 35 hits, there are total 56 non-CpG site mismatches, and 31 out of these 56 mismatches are A/G mismatch (55.4%). Among the rest 40 nonspecific captures, 15 contain two capture arms from one probe, 20 contacin only one capture arm.