Dinh/NOTES/2010-11-15

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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

Results[edit]