Noi/NOTES/2013-4-25: Difference between revisions
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* Jeff suggested not go over 15 cycles. I stopped reaction for 10ng UDG at 13 cycles and the rest at 15 cycles to make sure that I would see amplification. | * Jeff suggested not go over 15 cycles. I stopped reaction for 10ng UDG at 13 cycles and the rest at 15 cycles to make sure that I would see amplification. | ||
'''qPCR curve''' | '''qPCR curve''' | ||
[[File:20130425_lowinput_CGI.png| 650px]] | [[File:20130425_lowinput_CGI.png| 650px]] | ||
* The qPCR curve of NTC was lower than other samples. This was not as usual since I expected to see high background of adapters especially in NTC. | * The qPCR curve of NTC was lower than other samples. This was not as usual since I expected to see high background of adapters especially in NTC. | ||
* 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 2013-04-25 18hr 24min_lowinput_CGI_e.jpg| 520px]] [[File:ZhangLab_2 2013-04-25 18hr 16min_lowinput_CGI_e.jpg| 330px]] | [[File:ZhangLab_2 2013-04-25 18hr 24min_lowinput_CGI_e.jpg| 520px]] [[File:ZhangLab_2 2013-04-25 18hr 16min_lowinput_CGI_e.jpg| 330px]] | ||
S and L: with USER | |||
UDG = Uracil DNA glycosylase | |||
* Will add more info | * Will add more info |
Revision as of 03:38, 26 April 2013
- [Link to calendar]
- Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-4-24
- Received 12 positive control samples using the CGI protocol from Jeff (check details of his experiment in his page)
- Note from Jeff: I amplified e. coli gDNA using standard in tube MDA, with the addition of diluted dUTP at a very low concentration. I then purified, and diluted the amplicons to 1 ng and 10 ng. I then used USER/Uracil DNA glycosylase (UDG) to remove the dUTPs incorporated during MDA, and used Exo - klenow for nick translation and A tailing.
Sample list (4ul each) 2 10 ng, high USER conc. 2 10 ng, low USER conc. 2 1 ng, high USER conc. 2 1 ng, low USER conc. 2 1ng, UDG 2 10ng, UDG
- To process these samples
- Do adapter ligation, bead purification, and PCR
- Require at least 10X dilution of original volume (4ul --> need minimum ligation reaction 40ul)
- If the reaction works, we expected to see a smear from ~500 -1500bp
- Since the length of a smear is large, I would increase extension time during amplification to be longer (1min)
- Since DNA input is in the range of 1-10ng, I will used the same condition I tried on 2013_04_23 but using 50ul ligation reaction
Order of the tubes (need to check more info with Jeff) 1. 1ng high/low? USER short incubation 2. 1ng high/low? USER short incubation 3. 10ng high/low? USER short incubation 4. 10ng high/low? USER short incubation 5. 1ng high/low? USER long incubation 6. 1ng high/low? USER long incubation 7. 10ng high/low? USER long incubation 8. 10ng high/low? USER long incubation 9. 1ng UDG 10. 1ng UDG 11. 10ng UDG 12. 10ng UDG 13 . NTC
Adapter ligation
- Adapter ligation mix (Total 50ul) : Ligation reaction mix 49.2ul/reaction & 0.8ul of 3uM adapters (Diluted 30uM adapters 10X with cold 1X Stoffel buffer)
Ligation reaction mix | 1x rxn | 13x rxn mix |
KAPA 5X Ligation buffer | 10.00 | 130.00 |
KAPA DNA ligase | 5.00 | 65.00 |
H2O | 20.20 | 262.60 |
Total | 35.20 | 457.60 |
- Aliquot 35.2 ul of ligation reaction mix to each tube
- Transfer 4ul of sample to reaction tubes
- Rinse the tube with 10ul H2O and transfer all to reaction tube (total volume now is 49.2ul)
- Note: for NTC, I accidentally added too much H2O, so total of reaction of NTC was 60.8ul
- Add 0.8ul of 3uM adapters
- 20C for 15min (no shaking)
- Bead purification: 50ul 20% PEG/5M NaCl, wait 10min (no shaking), sit on magnet 5min, wash twice with fresh 80% EtOH, dry for 3 min
- Eluted with 15ul H2O (used all for PCR)
Amplification
Components | 1x rxn | 13.5 rxn mix |
adapter ligated DNA | 15.00 | 0.00 |
PCR_F(10uM) | 1.00 | 13.50 |
PCR_R.N2IndX(10uM) | 1.00 | 0.00 |
H2O | 8.00 | 108.00 |
2X KAPA SYBR MM | 25.00 | 337.50 |
Total volume | 50.00 | 675.00 |
- Aliquot 34ul, add 1ul of PCR_R.N2Indx, 15ul of adapter ligated DNA
Samples | Indx |
1. 1ng high/low? USER short incubation | PCR_R.N2Indx1 |
2. 1ng high/low? USER short incubation | PCR_R.N2Indx2 |
3. 10ng high/low? USER short incubation | PCR_R.N2Indx3 |
4. 10ng high/low? USER short incubation | PCR_R.N2Indx4 |
5. 1ng high/low? USER long incubation | PCR_R.N2Indx5 |
6. 1ng high/low? USER long incubation | PCR_R.N2Indx6 |
7. 10ng high/low? USER long incubation | PCR_R.N2Indx7 |
8. 10ng high/low? USER long incubation | PCR_R.N2Indx8 |
9. 1ng UDG | PCR_R.N2Indx21 |
10. 1ng UDG | PCR_R.N2Indx22 |
11. 10ng UDG | PCR_R.N2Indx23 |
12. 10ng UDG | PCR_R.N2Indx24 |
12. NTC | PCR_R.N2Indx24 |
Program (Eppendorf Realplex) 98C - 30s, (98C - 10s, 62C - 30s, 72C - 60s)x15, 72C - 2min, hold at 15C
- Jeff suggested not go over 15 cycles. I stopped reaction for 10ng UDG at 13 cycles and the rest at 15 cycles to make sure that I would see amplification.
qPCR curve File:20130425 lowinput CGI.png
- The qPCR curve of NTC was lower than other samples. This was not as usual since I expected to see high background of adapters especially in NTC.
- 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 2013-04-25 18hr 24min lowinput CGI e.jpg File:ZhangLab 2 2013-04-25 18hr 16min lowinput CGI e.jpg S and L: with USER UDG = Uracil DNA glycosylase
- Will add more info