Noi/NOTES/2013-4-30: Difference between revisions
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* 10ng USER/PNK : 12 cycles | * 10ng USER/PNK : 12 cycles | ||
* 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 2013-04-30 19hr 43min_CGI_qPCR.jpg| | [[File:ZhangLab_2 2013-04-30 19hr 43min_CGI_qPCR.jpg| 400px]] | ||
* From the gel image, using NEB loop adaptor can remove most of the background from adaptor. The library size was reduced since Jeff increased dUTP and got more amplification with smaller number of cycle. | * From the gel image, using NEB loop adaptor can remove most of the background from adaptor. The library size was reduced as expected since Jeff increased dUTP and got more amplification with smaller number of cycle. |
Revision as of 20:19, 1 May 2013
- [Link to calendar]
- Continued from:http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-4-25
- Got 8 samples processed by Jeff using CoRE fragmentation
- See Jeff's note for details of experiment
Sample list (4ul each) 3 1ng UDG/Endo IV 3 10ng UDG/Endo IV 2 10ng USER/PNK
- Manuals: [1]
- Some additional info for library prep (USER digestion and PCR): [2]
- Note: the manual didn't provide the concentration adapter and primers. I got the number from neb technical support
- Adapter and primer info
- NEBNext Adaptor
- Loop adapter containing U, need to be cleaved with USER after ligation.
- Conc. 15uM. Total volume 240ul. I aliquoted 25ul in 9 striptubes
- Since we used very little for ligtion (dilute 10X and add 1.67ul to each reaction for 1-10ng DNA input to get final conc. of adaptor in total 50ul reaction 0.05uM), this amount can be used to ligate ~1400 reactions
- NEBNext Universal PCR Primer
- Equivalent to PCR_F primer
- Conc. 25uM. Total volume 70ul. I dilute to 10uM in total volume 175ul
- NEbNext Index 1–12 Primers
- Conc. 25uM. Total volume 12ul. I dilute to 10uM in total volume 30ul and transferred to striptube for handling with multichannel pipette
- The sequences are different from PCR_R.N2Index (the same index but different in 3'- sequences. So if we used NEB adaptor, supposed to use reverse primer from NEB.
- NEBNext Adaptor
PCR_R.N2Ind1: 5'- CAAGCAGAAGACGGCATACGAGATCGTGATCTCGGCATTCCTGCTGAACCGCTCTT -3' NEBNext Index1: 5'- CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T -3' -s- = phosphorothioate bond * HP1 primer for read1 is the same since the NEBNext Universal PCR Primer is equivalent to PCR_F * Need to design new read2 primer (N2RevSeq2) --> NEBRevSeq2: 5'- GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT -3' (Tm = 74.4 C) * Need to design new index sequence read primer --> NEBIndSeq: 5'- GAAGAGCACACGTCTGAACTCCAGTCAC -3' (TM = 69.2 C)
- All sequences of adapter and primer from NEB kit will be updated on the Illunima GA/Oligo info in the main page
Adapter ligation
- I used the same concentration of adaptor as I did on April25, 2013
- Adapter ligation mix (Total 50ul) : Ligation reaction mix 48.33ul/reaction & 0.8ul of 3uM adapters (Dilute 15uM adapters 10X with H2O)
Adapter preparation
- Mix 2ul of 15uM loop adapter with 18ul H2O (final conc. 1.5uM).
Ligation reaction mix
Ligation reaction mix | 1x rxn | 9x rxn mix |
KAPA 5X Ligation buffer | 10.00 | 90.00 |
KAPA DNA ligase | 5.00 | 45.00 |
H2O | 19.33 | 173.97 |
Total | 34.33 | 308.97 |
- Aliquot 34.33 ul of ligation reaction mix to each tube
- Transfer 4ul of sample to reaction tubes
- As I mentioned before, if the sample is already in a clear tube, just add ligation reaction mix directly to fragmented DNA and adjust volume in ligation reaction mix
- I used 0.2 ml PCR Tube Strips, low-Profile tube (Biorad) to set up reaction
- Rinse the tube with 10ul H2O and transfer all to reaction tube (total volume now is 48.33ul)
- Add 1.67ul of 1.5uM loop adaptor
- 20C for 15min (no shaking)
- Add 1.5ul USER (come with the kit), 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, so might increase volume of USER to 2ul/ reaction or use 1.5ul with longer incubation (~30min) for the completion of USER digestion
- Bead purification: 50ul AmPure beads mix by pipetting 10 times, wait 10min (no shaking), sit on magnetic stand 5min, wash twice with freshly prepared 80% EtOH, dry for 3 min
- I was with 160ul of 80% EtOH since I used low profile tube.
- Eluted with 20ul H2O (resuspend and wait for 2min, used all for PCR)
Amplification
- I reduced extension time to 45 sec, and increase annealing temperature to 65C
Components | 1x rxn | 9.5 rxn mix |
adapter ligated DNA | 20.00 | 0.00 |
PCR_F (NEB, 10uM) | 1.00 | 9.50 |
PCR_R.N2IndX (NEB, 10uM) | 1.00 | 0.00 |
2X KAPA SYBR MM | 25.00 | 237.50 |
H2O | 3.00 | 28.50 |
Total volume | 50.00 | 475.00 |
- Aliquot 29ul, add 1ul of NEBNext Indx, 20ul of adapter ligated DNA
Samples | Indx |
1ng UDG/Endo IV -1 | NEBNext Index1 |
1ng UDG/Endo IV -2 | NEBNext Index2 |
1ng UDG/Endo IV -3 | NEBNext Index3 |
10ng UDG/Endo IV -1 | NEBNext Index4 |
10ng UDG/Endo IV -2 | NEBNext Index5 |
10ng UDG/Endo IV -3 | NEBNext Index6 |
10ng USER/PNK -1 | NEBNext Index7 |
10ng USER/PNK -2 | NEBNext Index8 |
NTC | NEBNext Index1 |
Program (Eppendorf Realplex) 98C - 30s, (98C - 10s, 65C - 30s, 72C - 45s)x15, 72C - 2min, hold at 15C
File:20130430 lowinput CGI.png * 1ng UDG/Endo IV : 13 cycles * 10ng UDG/Endo IV : 10 cycles * 10ng USER/PNK : 12 cycles
- 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 2013-04-30 19hr 43min CGI qPCR.jpg
- From the gel image, using NEB loop adaptor can remove most of the background from adaptor. The library size was reduced as expected since Jeff increased dUTP and got more amplification with smaller number of cycle.