Noi/NOTES/2013-5-22: Difference between revisions
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* [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']] | * [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']] | ||
= 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)) | * 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. | * Sample information should be noted by Eric. | ||
== Adaptor ligation == | |||
''' Reaction set up''' | |||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | |||
|- | |||
| Fragmented DNA||6.00 | | Fragmented DNA||6.00 | ||
|- | |- | ||
| 1.5uM | | 1.5uM NEBNext adaptor||1.67 | ||
|- | |- | ||
| Ligation reaction mix||42.33 | | Ligation reaction mix||42.33 | ||
Line 12: | Line 17: | ||
| Total||50.00 | | Total||50.00 | ||
|} | |} | ||
= | ''' NEBNext adaptor preparation ''' | ||
* I used 1.5uM adaptor prepared in the previous experiment | |||
* I diluted 15uM adaptor to 1.5uM (mix 20ul of 15uM NEBNext adaptor with 180ul H2O) and aliquoted 25ul into 8-striptubes and saved at -20C | |||
* The concentration of adaptor for ultra-low input DNA (<=10ng) in 50ul reaction is 50nM | |||
''' Ligation reaction mix ''' | |||
{| class="wikitable" style="text-align:center; | {| class="wikitable" style="text-align:center; | ||
| align="center" style="background:#f0f0f0;"|'''Ligation reaction mix''' | | align="center" style="background:#f0f0f0;"|'''Ligation reaction mix''' | ||
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* Aliquot 42.33 ul of ligation reaction mix to each tube containing 6ul of fragmented DNA | * 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. | * Add 1.67ul of 1.5uM NebNext loob adaptor. Mix well by pipetting 15-20 times and quick spin. | ||
* 25C for 10min | |||
* 25C for 10min | |||
* Add 2ul USER. Mix well by pipetting 15-20 times and quick spin. 37C for 15min | * 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 | ** 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) | * Eluted with 20ul H2O (resuspend and wait for 2min, used all for PCR) | ||
== Amplification == | |||
{| class="wikitable" style="text-align:center; | {| class="wikitable" style="text-align:center; | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' |
Revision as of 01:51, 23 May 2013
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.
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 used 1.5uM adaptor prepared in the previous experiment
- I diluted 15uM adaptor to 1.5uM (mix 20ul of 15uM NEBNext adaptor with 180ul H2O) and aliquoted 25ul into 8-striptubes and saved at -20C
- The concentration of adaptor for ultra-low input DNA (<=10ng) in 50ul reaction is 50nM
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
- 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 | 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 | 478.50 |
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