Revision as of 22:52, 22 May 2013 by >Noi
Ligation of CoRE fragmented DNA from Eric
- Got 16 samples from Eric for ligation using NEBNext adaptor. (Samples were already in 0.2 ml PCR Tube Strips, low-Profile tube (Biorad))
- Sample information should be noted by Eric.
Fragmented DNA |
6.00
|
1.5uM loop adaptor |
1.67
|
Ligation reaction mix |
42.33
|
Total |
50.00
|
Ligation reaction mix
Ligation reaction mix
|
1x rxn
|
16x rxn mix
|
2X KAPA Rapid Ligation Buffer |
25.00 |
400.00
|
KAPA Rapid T4 DNA Ligase |
2.00 |
32.00
|
H2O |
15.33 |
245.28
|
Total |
42.33 |
677.28
|
- 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 (no shaking).
- 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 (no shaking)
- 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
|
16.5 rxn mix
|
adapter ligated DNA |
20.00 |
0.00
|
PCR_F (NEB, 10uM) |
1.00 |
16.50
|
PCR_R.N2IndX (NEB, 10uM) |
1.00 |
0.00
|
2X KAPA SYBR MM |
25.00 |
412.50
|
H2O |
3.00 |
49.50
|
Total volume |
50.00 |
825.00
|
Samples
|
Indx
|
Samples
|
Indx
|
#1 |
NEBNext Index1 |
#9 |
NEBNext Index1
|
#2 |
NEBNext Index2 |
#10 |
NEBNext Index2
|
#3 |
NEBNext Index3 |
#11 |
NEBNext Index3
|
#4 |
NEBNext Index4 |
#12 |
NEBNext Index4
|
#5 |
NEBNext Index5 |
#13 |
NEBNext Index5
|
#6 |
NEBNext Index6 |
#14 |
NEBNext Index6
|
#7 |
NEBNext Index7 |
#15 |
NEBNext Index7
|
#8 |
NEBNext Index8 |
#16 |
NEBNext Index8
|
- Aliquot 29ul, add 1ul of NEBNext Indx, 20ul of adapter ligated DNA
Program (Eppendorf Realplex)
98C - 30s, (98C - 10s, 65C - 30s, 72C - 45s)x15, 72C - 2min, hold at 15C
I stopped the reaction at 12 cycles
File:20130522 qPCR CoRE.png
- Eric should provide the sample info.
- 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 2013-05-22 13hr 33min gel1 1-8.jpg File:ZhangLab 2 2013-05-22 13hr 35min gel2 9-18.jpg