Dinh/NOTES/2010-11-15: Difference between revisions
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**There is no need of doing blue-white screening using X-gal, because the pBlunt plasmid has selfligation-proof design. | **There is no need of doing blue-white screening using X-gal, because the pBlunt plasmid has selfligation-proof design. | ||
*Incubate plates at 37C for about 20hr. <6pm 11/18/10 -> 4pm 11/19/10> | *Incubate plates at 37C for about 20hr. <6pm 11/18/10 -> 4pm 11/19/10> | ||
'''Pick up 10 colonies per sample''' | |||
*Prepare LB medium with 10% glycerol and 35ug/ml Kanamycin. | |||
*200ul per well | |||
[[File:Bis-PCR Progeria - plate layout.png]] | |||
==Results== | ==Results== | ||
Latest revision as of 01:20, 20 November 2010
Bisulfite sequencing for Oct4, Nanog, and LMNA promoter methylation[edit]
- Measuring the methylation at several CpGs in control genes: Oct4, Nanog, and LmnA.
- The data can be used as control of methylation at pluripotent genes
- As requestd by Guanghui, I will primers previously published in (Mol Biol Cell. 2007 May; 18(5): 1543–1553. doi: 10.1091/mbc.E07-01-0029.)
Primers[edit]
Name | Sequence | Length |
bs-Oct4-5-F | AATAGATTTTGAAGGGGAGTTTAGG | 184bp |
bs-Oct4-5-R | TTCCTCCTTCCTCTAAAAAACTCA | chr6:31,140,029-31,140,212 |
bs-Nanog2-F | GAGTTAAAGAGTTTTGTTTTTAAAAATTAT | 294bp |
bs-Nanog2-R | TCCCAAATCTAATAATTTATCATATCTTTC | chr12:7,940,792-7,941,084 |
bs-LMNA-F | GAAGGGTGAGTTATATTGATGGGTAT | 370 |
bs-LMNA-R | ACTCTTAAAAAAACAATCCCAAATC | chr1:156,084,071-156,084,439 |
PCR[edit]
biscvt DNA (Conc by QUBIT):
#1 HGPS72 - 202.57 ng/ul #2 H9 <resolve H9 vs BJ mix-up> - 47.21 ng/ul #3 BJ fib - 46.01 ng/ul #4 BJiPS #3 - 95.62 ng/ul #5 HGPSiPS #4 - 48.93 ng/ul #6 HGPSiPS #2 - 65.92 ng/ul #7 BJiPS #6 - 26.06 ng/ul Template (10ng/ul): 1ul F primer (25uM) : 1ul R primer (25uM) : 1ul H20 : 17ul 2xiQ MM : 20ul ----------------------- 40ul
Samples Setup: N1 N2 N3 N4 N5 NTC L1 L2 L3 L4 L5 NTC O1 O2 O3 O4 O5 O6 O7 NTC
O - Oct4, L - LmnA, N - Nanog
Program: 96C 3min 95C 30s 58C 45s 72C 45s Go to 2 for 34x 72C 5min 15C forever
Purify by 1x Ampure beads.
File:BisPCR progeria first round rt pcr.PNG File:ZhangLab 2 2010-11-16 bis PCR progeria 2.jpg
- Quantification by Nanodrop
- Purification by E-Gel 2%
File:ZhangLab 2 2010-11-16 E-Gel ss bisPCR amplicons.jpg
- Purification by 1x Ampure Beads
Blunt-vector ligation[edit]
Quick blunting Kit
x 18rxn mix DNA 38ul - 10X blunting buffer. 5ul 90ul dNTP mix (1mM) 5ul 90ul Blunting Enzyme Mix 2ul 36 ------------------------------------------ - aliquot 12ul of reaction mix into PCR tube, - add 38ul of DNA - incubate at room temperature for 30 minutes, purify with 0.7x AMPure beads, elute with 40ul EB.
PAGE Gel File:ZhangLab 2 2010-11-17 bisPCR progeria blunt ended DNA.jpg
Reamplification with Phusion HF for 10 cycles File:BisPCR progeria second round rt pcr.PNG
- Purification with 0.7x Ampure beads
- PAGE Gel
File:ZhangLab 2 2010-11-17 bisPCR progeria re-amped ended amplicons.jpg
- Concentrate O1-O7 using concentrator.
- Invitrogen Zero-blunt PCR Cloning kit (Invitrogen)
- Set up ligation reaction in PCR tubes on cold block:
Sam's protocol (Invitrogen manual protocol) Mix O Mix N Mix L PCR product of seq. lib 2.0 2.3 0.8 10X Ligation buffer 1.0 1.0 1.0 pCR-Blunt (25ng/uL) 0.5 0.5 0.5 RT-PCR grade H2O 5.5 5.2 6.7 (Red cap)T4 DNA ligase (4U/uL) 1.0 1.0 1.0 ---------------------------------------------------------------------- 10.0 10.0 10.0
- Incubate at 16C for 1hr using PCR thermocycler. Perform the transfection using the ligation product as soon as possible.
Heat-shock transfection[edit]
- Equilibrate water bath to 42C.
- Bring the SOC medium, LB plates (w/ antibiotics - Kanamycin 50ug/mL) to RT for 30min.
- Thaw the TOP10 E.coli competent cell vial on ice.
- Transfer 1uL ligation reactions into the vial of competent cells. Mix by gentle stiring using tips. (Mixing by repeat pipetting is prohibited in the protocol).
- Set up competent cell vials in the foam-board holder (for water bath). Incubate the vials on ice for 30 mins.
- Adjust the timer: set up 30sec and 2min.
- Heat shock the cells for 30 seconds at 42 C without shaking. Immediately transfer the vials on ice for 2min.
- Add 250ul of SOC medium into each vial (drop the SOC solution slowly throw the tube wall).
- Shake the vials horizontally at 37C for 1 hour using vortex on 3rd floor at the lowest speed.
- During the 1hr incubation.
- After 1hr shaking, plate 50uL from each transformation vial on agar plates (w/ Kanamycine).
- There is no need of doing blue-white screening using X-gal, because the pBlunt plasmid has selfligation-proof design.
- Incubate plates at 37C for about 20hr. <6pm 11/18/10 -> 4pm 11/19/10>
Pick up 10 colonies per sample
- Prepare LB medium with 10% glycerol and 35ug/ml Kanamycin.
- 200ul per well
File:Bis-PCR Progeria - plate layout.png