Revision as of 03:35, 26 April 2013 by >Noi
- [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