Noi/NOTES/2014-12-16: Difference between revisions
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[[File:ZhangLab_2 2014-12-21 17hr 59min_PhusionFlash-quickPCR-LMS-WGBS-5ul.jpg| 450px]] [[File:ZhangLab_2 2014-12-21 17hr 59min_KAPAHiFi-quickPCR-LMS-WGBS-5ul.jpg| 450px]] | [[File:ZhangLab_2 2014-12-21 17hr 59min_PhusionFlash-quickPCR-LMS-WGBS-5ul.jpg| 450px]] [[File:ZhangLab_2 2014-12-21 17hr 59min_KAPAHiFi-quickPCR-LMS-WGBS-5ul.jpg| 450px]] | ||
* The pattern of PCR products was consistent between the two enzymes. However, Phusion Flash Hi-Fidelity showed stronger intensity, so I will keep using this enzyme for the future. This should be note that a lower signal from KAPA HiFi Hotstart ReadyMix might be due to too short denaturation. However, I had no time to repeat experiment. | * The pattern of PCR products was consistent between the two enzymes. However, Phusion Flash Hi-Fidelity showed stronger intensity, so I will keep using this enzyme for the future. This should be note that a lower signal from KAPA HiFi Hotstart ReadyMix might be due to too short denaturation. However, I had no time to repeat experiment. | ||
* I continued to amplified the rest of captured DNA in large volume (200ul --> split 2X of 100ul reaction) for | * I continued to amplified the rest of captured DNA in large volume (200ul --> split 2X of 100ul reaction) for 20 cycles using Phusion Flash Hi-Fidelity. |
Revision as of 08:32, 13 January 2015
LMS (MONOD V5A) BSPP capture on WGBS libraries prepared from cfDNA of normal control and cancer patient
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Continued from 2014-12-05
- Conditions:
- Probe set: MONOD V5 set A (LMS)
- Probe:target ratio: 150:1 for LMS probe set.
- Please note that I used very high amount of WGBS libraries for BSPP capture up to 600ng. This resulted in a very high amount of BSPP required for capture. I therefor reduced probe to target ratio from 200:1 to 125:1. The main reason is I do not have enough LMS BSPP to capture 50 samples with 600ng DNA each. For experiment I did for Illumina (Li Liu), I used 400ng of WGBS libraries, so I had enough probes to capture that amount. In order to be able to capture all unique molecule of ctDNA, I tried to increase WGBS libraries as much as possible. Ideally, Dr. Zhang wanted 1ug for capture.
- Sample:
- WGBS libraries of ctDNA from cancer patients prepared by KAPA Hyper Prep Kit 600ng.
- WGBS libraries of cfDNA from normal control (re-amplified) prepare by using RRBS protocol but skipping DNA digestion/fragmentation 600ng
Probe calculation
Probe set Probe size Probe:target ratio LMS 73,574 150:1 From 2014-12-08 exp., 400ng required 108ng (200:1). So 600ng (150:1) requires ~100ng of LMS probes
- Since the probe concentration a little low (8.25ng/ul), I will set up capture reaction in 300ul.
NOTE: WHAT ARE DIFFERENT FROM EXP. on 2014-12-08 (ILLUMINA, for Li Liu)
- Template 600ng instead of 400ng
- Probe: target ratio is 125:1 instead of 200:1
- Capture reaction 30ul instead of 25ul
Precedures
- I took 600ng of WGBS libraries in 1.5mL LoBind tube. Rationale: total volume of 600ng of WGBS libraries is too large to fit in 30ul capture reaction.
- Speedvac at 60C for ~20-30min to dry up DNA in the tube.
- Resuspend each tube with 14.88ul H2O which is the maximum volume of WGBS libraries to fit in 30ul capture reaction.
- Make sure that we resuspend DNA on the bottom of the tube very well.
- Transfer 14.88ul of DNA to 95-well plate
- I re-organized the location of each sample on the well based on sample ID (small - large)
- Aliquot 15.12ul of Probe & Buffer Mix in below table to each well
- Total volume is 30ul.
- Mix well, seal with plastic seal and spin down the plate
- Layer with 30ul mineral oil
- Seal with Aluminum seal and spin down the plate again.
- Incubate the plate on thermocycler following program below
Sample | Conc. (ng/ul) | Volume for 600ng (ul) |
NC-1 | 13.73 | 43.69 |
NC-2 | 16.00 | 37.50 |
NC-3 | 16.93 | 35.43 |
NC-5 | 13.07 | 45.92 |
NC-6 | 15.33 | 39.13 |
NC-7 | 13.73 | 43.69 |
NC-8 | 15.87 | 37.82 |
NC-9 | 16.53 | 36.29 |
NC-12 | 14.80 | 40.54 |
NC-13 | 13.19 | 45.50 |
NC-14 | 14.93 | 40.18 |
NC-15 | 13.60 | 44.12 |
NC-16 | 13.87 | 43.27 |
NC-17 | 13.47 | 44.55 |
NC-18 | 14.67 | 40.91 |
NC-19 | 13.47 | 44.55 |
NC-20 | 12.79 | 46.92 |
NC-21 | 12.31 | 48.75 |
NC-22 | 16.00 | 37.50 |
NC-23 | 13.60 | 44.12 |
NC-25 | 11.97 | 50.11 |
NC-27 | 14.67 | 40.91 |
NC-29 | 12.96 | 46.30 |
NC-30 | 15.47 | 38.79 |
Sample | Conc. (ng/ul) | Volume for 600ng (ul) |
PCP-9 | 26.13 | 22.96 |
PCP-4 | 21.73 | 27.61 |
PCP-8 | 19.07 | 31.47 |
PCP-6 | 20.13 | 29.80 |
PCP-7 | 22.80 | 26.32 |
PCP-2 | 22.13 | 27.11 |
PCP-3 | 21.47 | 27.95 |
PCP-5 | 21.47 | 27.95 |
6P-1 | 26.00 | 23.08 |
6P-8 | 24.80 | 24.19 |
6P-10 | 22.27 | 26.95 |
6P-9 | 20.27 | 29.61 |
6P-5 | 24.67 | 24.32 |
6P-2 | 19.60 | 30.61 |
6P-3 | 19.87 | 30.20 |
6P-4 | 19.20 | 31.25 |
7P-8 | 22.80 | 26.32 |
7P-3 | 19.07 | 31.47 |
7P-10 | 25.33 | 23.68 |
7P-7 | 29.60 | 20.27 |
7P-6 | 26.93 | 22.28 |
7P-1 | 14.67 | 40.91 |
7P-2 | 19.73 | 30.41 |
7P-5 | 18.00 | 33.33 |
- In 96-well plate, I re-organized the sample ID from small to large like from PCP2, PCP-3, PCP4,....
Probe & Buffer Mix
Components | 1X | 51X |
MONOD V5A (LMS) 8.25ng/ul | 12.12 | 618.18 |
10X Ampligase Buffer | 3.00 | 153.00 |
WGBS library (600ng) | 14.88 | |
Total | 30.00 |
Program -> 95c 5min -> cool down to 55C at 0.02C/sec -> 55C 23h -> add 2.5ul PLN mix (20%v/v Hemo Klentaq; 0.5U/ul AmpLigase; 100uM dNTP) --> No need to change program while adding PLN mix (keep the tube at 55C all the time) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 5min -> 10C hold. --> Press proceed once before adding ExoI/III and waiting until temperature down to 37C before adding ExoI/III
PLN (Polymerase/Ligase/ dNTP) mix solution Mix
Components | Stock conc. | Unit | Final conc. | Unit | Prepare volume (ul), total 150ul |
Hemo KlenTaq | 100 | % | 20 | % | 30.00 |
AmpLigase | 5 | U/ul | 0.5 | U/ul | 15.00 |
dNTP | 1 | mM | 100 | uM | 15.00 |
10x AmpLigase Buffer | 10 | x | 1 | x | 15.00 |
H2O | 75.00 | ||||
Total | 150.00 |
- Prepare PLN solution mix as follows: 20% (v/v) Hemo KlenTaq, 0.5 unit/μL Ampligase, 100 μM of dNTP mix, and 1x Ampligase Buffer.
- Aliquot 18.5ul of PLN mix to 8-tube strip and add 2.5 μL of KLN solution mix to each well with 10 μL multichannel pipette. Make sure that PLN mix is added under the mineral oil layer.
- Swirl pipette 10 times to mix the capture reaction without moving the plate from thermal cycler.
- Re-seal a 96-well plate with aluminum-seal.
- Continue to incubate at 55 ºC for 20 hours.
- Denature DNA at 94 ºC for 2 minutes.
Exonuclease digestion
- Prepare exonuclease I/III mix by mixing exonuclease I (20 units/μL) and exonuclease III (200 units/μL) in 1:1 ratio and aliquot equal volume to 8-tube strip
- Add 2 μL of exonuclease I/III mix to each well with multichannel pipette
- Mix the reaction by swirling pipette around the well 10 times.
- Incubate reaction at 37 ºC for 2 hours.
- Heat inactivate exonuclease I/III at 94 ºC for 5 minutes.
- Continue to qPCR without any purification or store at 4C
Amplification
- I normally do a quick test PCR in small volume 20ul with 10% v/v of captured DNA. This to monitor PCR cycle number and check if we get the right amplified product by running PCR product in 6% TBE gel without bead/colum purification. If the experiment has been optimized for a certain probe set, may not need to do quick qPCR check. This time I want to compare between the two enzymes, Phusion Flash High-Fidelity MM and KAPA HiFi Hotstart ReadyMix.
Quick PCR
- I randomly picked few samples from normal controls and cancer patient samples for a quick test PCR, including NC-1, NC-2, NC-3 NC-4, PCP-2, PCP-3, PCP-4, PCP-5, ^T1 from previous exp. as positive control and NTC.
Components | Stock conc. | Final conc. In PCR | 1x rxn | 13x rxn |
Captured template | 100% | 10% | 2.00 | 0.00 |
10uM AmpF6.4Sol | 10uM | 0.2uM | 0.40 | 5.20 |
10uM AmpR6.3.IndX 8 | 10uM | 0.2uM | 0.40 | 5.20 |
2X Phusion Flash Hi-Fidelity MM or KAPA HiFi Hotstart ReadyMix | 2X | 1X | 10.00 | 130.00 |
50X SYBR | 50X | 0.4X | 0.16 | 2.08 |
H2O | 7.04 | 91.52 | ||
Total | 20.00 |
- - Aliquot 18ul + 2ul of captured DNA template
- Note: In PCR, I included one of positive control (PTC) of captured templates from gDNA of tumor tissue sample to compare the size of amplified amplicons.
- - 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x10-15 cycles -> 72C 3min
- Note that I just realized that KAPA HiFi Hotstart ReadyMix requires 2-3min for hot start activation. I paused the machine for 1 min a9 98C during denaturing step. I was not sure if this will result in not-fully active DNA polymerase for KAPA.
- - In this experiment, we stopped at 19 cycles
- - Loaded 5ul of PCR product in 6% TBE gel without purification (Run at 250 volts in 0.5X TBE buffer for 25min)
File:ZhangLab 2 2014-12-21 17hr 59min PhusionFlash-quickPCR-LMS-WGBS-5ul.jpg File:ZhangLab 2 2014-12-21 17hr 59min KAPAHiFi-quickPCR-LMS-WGBS-5ul.jpg
- The pattern of PCR products was consistent between the two enzymes. However, Phusion Flash Hi-Fidelity showed stronger intensity, so I will keep using this enzyme for the future. This should be note that a lower signal from KAPA HiFi Hotstart ReadyMix might be due to too short denaturation. However, I had no time to repeat experiment.
- I continued to amplified the rest of captured DNA in large volume (200ul --> split 2X of 100ul reaction) for 20 cycles using Phusion Flash Hi-Fidelity.