Noi/NOTES/2013-5-24: Difference between revisions
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I plotted qPCR curves separately according to the gel loading order because I can't clearly observe amplification curve of each sample by plotting all 24 samples together. | I plotted qPCR curves separately according to the gel loading order because I can't clearly observe amplification curve of each sample by plotting all 24 samples together. | ||
[[File:2013-05-24_Eric.qPCR_1-8.png| | [[File:2013-05-24_Eric.qPCR_1-8.png| 325px]] [[File:2013-05-24_Eric.qPCR_9-16.png| 325px]] [[File:2013-05-24_Eric.qPCR_17-24.png| 325px]] | ||
* PAGE analysis to verify the PCR products by loading 5 ul of PCR product without bead purification (1/10 of total volume) | * PAGE analysis to verify the PCR products by loading 5 ul of PCR product without bead purification (1/10 of total volume) | ||
[[File:ZhangLab_2 2013-05-24 15hr 41min_1-8.jpg| 320px]] [[File:ZhangLab_2 2013-05-24 15hr 43min_9-16.jpg| 325px]] [[File:ZhangLab_2 2013-05-24 15hr 46min_17-24_e.jpg| 325px]] | [[File:ZhangLab_2 2013-05-24 15hr 41min_1-8.jpg| 320px]] [[File:ZhangLab_2 2013-05-24 15hr 43min_9-16.jpg| 325px]] [[File:ZhangLab_2 2013-05-24 15hr 46min_17-24_e.jpg| 325px]] | ||
* All samples worked as expected since Eric did qPCR to verify the yield of MDA. | * All samples worked as expected since Eric did qPCR to verify the yield of MDA. | ||
* The average size of libraries looked smaller than the ones on May22 | * The average size of libraries looked smaller than the ones on May22 |
Revision as of 01:11, 25 May 2013
Adaptor ligation of CoRE fragmented DNA (Eric's May 23, 2013 experiment)
- Got 24 samples from Eric for ligation using NEBNext adaptor.
- MDA and CoRE fragmentation were performed by Eric and documented on Eric's note on May23, 2013: [[1]]
- All procedures of adaptor ligation and amplification were the same as my experiment on May22, 2013
Adaptor ligation
Reaction set up
Components | Volume (ul) |
Fragmented DNA | 6.00 |
1.5uM NEBNext adaptor | 1.67 |
Ligation reaction mix | 42.33 |
Total | 50.00 |
NEBNext adaptor preparation
- I diluted a new batch of adaptor by diluting 15uM adaptor to 1.5uM (mix 20ul of 15uM NEBNext adaptor with 180ul H2O) and aliquoted 25ul into 8-striptubes and saved at -20C after used.
Ligation reaction mix
Ligation reaction mix | 1x rxn | 24x rxn mix |
2X KAPA Rapid Ligation Buffer | 25.00 | 600.00 |
KAPA Rapid T4 DNA Ligase | 2.00 | 48.00 |
H2O | 15.33 | 367.92 |
Total | 42.33 | 1,015.92 |
- Aliquot 42.33 ul of ligation reaction mix to each tube containing 6ul of fragmented DNA
- Add 1.67ul of 1.5uM NebNext loob adaptor. Mix well by pipetting 15-20 times and quick spin.
- 25C for 10min
- Add 2ul USER. Mix well by pipetting 15-20 times and quick spin. 37C for 15min
- For the library prep using NEBNext® DNA Library and this loop adaptor, they used 3ul USER in total volume 50ul or 83.5ul, 37 C for 15 min. I used a little less
Bead purification
- 50ul AmPure beads mix by pipetting 10 times
- Wait 10min
- Sit on magnetic stand 5min
- Wash twice with freshly prepared 80% EtOH (after adding 80% EtOH, I wait for 30sec before pipette up and down few times and discard spnt)
- I wash with 160ul of 80% EtOH since I used low profile tube.
- Dry the beads for 3 min
- Eluted with 20ul H2O (resuspend and wait for 2min, used all for PCR)
Amplification
Components | 1x rxn | 25 rxn mix |
adapter ligated DNA | 20.00 | 0.00 |
PCR_F (NEB, 10uM) | 1.00 | 25.00 |
NEBNext.IndX (NEB, 10uM) | 1.00 | 0.00 |
2X KAPA SYBR MM | 25.00 | 625.00 |
H2O | 3.00 | 75.00 |
Total volume | 50.00 | 725.00 |
- Aliquot 29ul, add 1ul of NEBNext Indx, 20ul of adapter ligated DNA
Samples | Indx | Samples | Indx |
#1 | NEBNext Index1 | #13 | NEBNext Index1 |
#2 | NEBNext Index2 | #14 | NEBNext Index2 |
#3 | NEBNext Index3 | #15 | NEBNext Index3 |
#4 | NEBNext Index4 | #16 | NEBNext Index4 |
#5 | NEBNext Index5 | #17 | NEBNext Index5 |
#6 | NEBNext Index6 | #18 | NEBNext Index6 |
#7 | NEBNext Index7 | #19 | NEBNext Index7 |
#8 | NEBNext Index8 | #20 | NEBNext Index8 |
#9 | NEBNext Index9 | #21 | NEBNext Index9 |
#10 | NEBNext Index10 | #22 | NEBNext Index10 |
#11 | NEBNext Index11 | #23 | NEBNext Index11 |
#12 | NEBNext Index12 | #24 | NEBNext Index12 |
Program (Eppendorf Realplex) 98C - 30s, (98C - 10s, 65C - 30s, 72C - 45s)x15, 72C - 2min, hold at 15C I stopped the reaction at 11 cycles
I plotted qPCR curves separately according to the gel loading order because I can't clearly observe amplification curve of each sample by plotting all 24 samples together. File:2013-05-24 Eric.qPCR 1-8.png File:2013-05-24 Eric.qPCR 9-16.png File:2013-05-24 Eric.qPCR 17-24.png
- PAGE analysis to verify the PCR products by loading 5 ul of PCR product without bead purification (1/10 of total volume)
File:ZhangLab 2 2013-05-24 15hr 41min 1-8.jpg File:ZhangLab 2 2013-05-24 15hr 43min 9-16.jpg File:ZhangLab 2 2013-05-24 15hr 46min 17-24 e.jpg
- All samples worked as expected since Eric did qPCR to verify the yield of MDA.
- The average size of libraries looked smaller than the ones on May22