Kun:LabNotes/Haplotyping/2011-5-12: Difference between revisions

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   [[Image:In-Gel-biscvt-MDA-tagmentation-PCR-AmpCurve.png|300px]][[Image:In-Gel-biscvt-MDA-tagmentation-PCR-13May2011.png|300px]]
   [[Image:In-Gel-biscvt-MDA-tagmentation-PCR-AmpCurve.png|300px]][[Image:In-Gel-biscvt-MDA-tagmentation-PCR-13May2011.png|300px]]
*Asked Alice to perform AmpPure bead purification, then 2nd PCR to add barcodes and sequencing adaptors.
*Asked Alice to perform AmpPure bead purification, then 2nd PCR to add barcodes and sequencing adaptors.
===Discussions====
===Discussions===
#I terminated the PCR reactions at the end of 22th cycle when the amplification curves for the 7 reactions with gel came up whereas the NTC was still almost flat. This is a good indication that tagmentation worked to some extend. Based on the PAGE gel, there are faint smears in the 7 reactions. However they are not strong enough, which most likely because the PCR reactions were terminated perhaps 1-2 cycles earlier. Overall this seems to be promising enough to proceed.
#I terminated the PCR reactions at the end of 22th cycle when the amplification curves for the 7 reactions with gel came up whereas the NTC was still almost flat. This is a good indication that tagmentation worked to some extend. Based on the PAGE gel, there are faint smears in the 7 reactions. However they are not strong enough, which most likely because the PCR reactions were terminated perhaps 1-2 cycles earlier. Overall this seems to be promising enough to proceed.
#There are a few steps that could be improved in the future experiments.
#There are a few steps that could be improved in the future experiments.

Revision as of 05:25, 14 May 2011

Methylation haplotyping on small number of cells

Idea

  • Directly lyse a small number of cells, remove all proteins, denature the gDNA, and trap the ssDNA in polyacrylamide gel.
  • Perform bisulfite conversion, followed by limited MDA and tagmentation all directly within the gel.
  • Dissect the gel into a number of small pieces, such that each piece contains less than haploid genome, followed by barcoded PCR in tubes.
  • The PCR amplicons are pooled for sequencing.

Experiment protocol

  • Make dilution of GM20431 cells to 1 cell/ul.
  • Mix 10ul of cell solution with 10ul Cell Lysis Buffer(20M EDTA, 10mM Tris.HCl , 200mM NaCl, 0.2% Triton X-100, 0.2AU/ml Qiagen Protease), incubate at 37C for 30min, 75C for 15min. Slowly mix the cell lysate with a P20 pipette for 10 times (to break down chromosomes).
  • Prepare gel mix:
      H2O	           10ul
      ABD	            8ul
      30% BSA            0.3ul  
      Cell lysate         20ul
      5% TEMED           0.8ul
   Heat at 94C for 5min, immediately transfer to ice
   Add 5% APS 	  0.8ul 
  • Add 18ul each to two slides, wait ~15 minutes for the gel to polymerize.
  • Wash the slides with ddH2O for >5min.
  • Place a frameseal chamber on each slide, add 120 ul CT Conversion Reagent (Zymo EZ DNA Methylation Direct Kit), seal the chamber.
  • Incubate at 95C 4min -> 64C 4h -> 4C hold.
  Roughly half of the CT conversion reagent evaporated in less than 2 hours despite my best efforts in sealing the chamber.
  So I removed the chambers, put new ones and added new CT Conversion Reagent. 
  95C 1min -> 64C 2h 15min -> 4C hold.
  • Wash the slides twice with ddH2O for >5min.
  • Add ~50ul of M-desulphonation buffer onto the gel and let it stand at RT for 15min.
  • Wash the slides twice with ddH2O for >5min.
  • Set up limited MDA reaction:
     10x RepliPhi Buffer       4.0ul
     1mM N6 primer             2.0ul
     25mM dNTP                 0.4ul
     2X SYBR Green I           2.0ul
     RepliPhi Phi29(100U/ul)   2.0ul
     Exo-minus Klenow (5U/ul)  1.0ul
     H2O                      28.0ul
     30% BSA                   0.7ul
   After adding MDA mix to the gel and cover with coverslips, carefully pipette mineral oil to cover the edges of coverslips.
   Incubate one slide at 30C for 30 min, the other slide at 30C for 1h, head inactivation at 65C for 10min
  • Wash the slide with ddH2O for > 5min, air-dry inside PCR hood.
  • Prepare tagmentation mix:
   Dilute the enzyme: 1:5     
     5x LMW Buffer             8ul
     diluted enzyme            4ul
     H2O                      28ul
   Add 20ul to each gel, cover with a coverslip, seal the edges with mineral oil, incubate at 55C for 10min
  • Wash the Wash the slide with ddH2O for > 5min, use a clean scalpel to cut the gel into 12 slices, transfer one into each PCR tube.
  • Set up PCR reaction:
                                      x 12
     KAPA QPCR mix            25ul
     Orange Primer (10uM)      1ul
     Blue Primer (10uM)        1ul
     Bst Pol (5U/ul)         0.5ul
     H2O                      23ul
     65C 15min -> 95C 30 sec -> (95C 10sec -> 58C 30 sec -> 72 1min) x 22 -> 72C 3min. 
    Monitor the reactions on a real-time thermal cycler and terminate them before the curves reach saturation.
  • If the amplification curves are good, check the amplicon size with 6% TBE gel.
  File:In-Gel-biscvt-MDA-tagmentation-PCR-Plate.pngFile:In-Gel-biscvt-MDA-tagmentation-PCR-legend.png
  File:In-Gel-biscvt-MDA-tagmentation-PCR-AmpCurve.pngFile:In-Gel-biscvt-MDA-tagmentation-PCR-13May2011.png
  • Asked Alice to perform AmpPure bead purification, then 2nd PCR to add barcodes and sequencing adaptors.

Discussions

  1. I terminated the PCR reactions at the end of 22th cycle when the amplification curves for the 7 reactions with gel came up whereas the NTC was still almost flat. This is a good indication that tagmentation worked to some extend. Based on the PAGE gel, there are faint smears in the 7 reactions. However they are not strong enough, which most likely because the PCR reactions were terminated perhaps 1-2 cycles earlier. Overall this seems to be promising enough to proceed.
  2. There are a few steps that could be improved in the future experiments.
    1. I wasn't very confident on the quality of cell lysis, because Alice got much better results with the other buffer using NP40. I did add fresh protease into this lysis buffer, and also used the buffer to lyse ~20,000 cells and checked with microscope. The cells seemed to be lysed, but I still think it worth trying the NP40-based buffer next time.
    2. On-gel bisulfite conversion is tricky, because it's hard to fill the FrameSeal chamber completely and prevent leaking during the high-temperature incubation. It is possible that the conversion rate is better in some area of the gel than the other. Covering the gel with coverslips and seal with mineral oil seems to be a better option. Definitely worth trying next time.
    3. I check the gel with microscrope at SYBG channel after MDA, but didn't see any signal except for some debris. I wasn't sure whether the MDA was working or not. However, if the amplification occurred evenly across the gel, I would not see anything either. Since I was able to get some amplicons after tagmentation/PCR, it's likely that MDA did work to some extend.
    4. There were a few bubble on the gel when I did in-gel tagmentation. Some small areas of the gel was damaged after the previous steps, such that the coverslip couldn't sit flat on the gel. One solution is to be more careful and try not to damage the gel. Another idea is to increase the volume of tagmentation reaction mix from 20ul to 25-30ul.
    5. Cutting gel into small piece and collecting the pieces individually is a little bit tricky. It's important not to do it when the gel is too dry. Transferring little gel pieces into PCR tubes is not easy either. I ended up pipetting 5ul of clean water to the scalpel tip that carries the gel piece, then transferring the water (with gel piece inside) to PCR tubes with the help of a P20 pipette.