AlanFung:LabNotes/EZ/2009-3-25
EZ DNA Methylation Direct - 10 GM20431 cells[edit]
Objective[edit]
- Confirmation of the bisulfite conversion of GM20431 for 10 cells
- Elute DNA with 8uL elution buffer
- Use all 8uL dna as template for one pair of primer
- Reduce washing step from two times to one
- For one set (3x10cells- one washing before elution)
- For the other set (2x10cells-two washing before elution
Samples & Materials[edit]
- GM20431 P20 cells
- EZ DNA Methylation Direct Kit 03/11/09
- Primers - From IDT
-------------------------------------------------- 0.1_F_chr22_31384238GTGAATAGGTTAAGTGAGGTAGAAG 0.1_R_chr22_31384238AAAAAAATCAAACACCAACTATAAA 0.8_F_chr21_39672131AAAATATTGGGATTATAGGTATGAGT 0.8_R_chr21_39672131AACTTCTAAACTAACCAAAACAAAA 0.9_F_chr8_119031762TTATAGTTTGGGTGATAGAGTAAGATT 0.9_R_chr8_119031762AAACCCTAAACAAAATACTCAATATAA --------------------------------------------------
- Creating 100uM primer
0.1_F_chr22 27.9nmol RNAse free H2O 279uL
0.1_R_chr22 31.8nmol RNAse free H2O 318uL
0.8_F_chr21 31.30nmol RNAse free H2O 313uL
0.8_R_chr21 31.70nmol RNAse free H2O 317uL
0.9_F_chr8 30.10nmol RNAse free H2O 301uL
0.9_R_chr8 29.30nmol RNAse free H2O 293uL
- Making 3.3uM primer working solution
Dilute 33uL 100uM primer with 967uL RNAse free H2O
- Jurkat gDNA (100ug/mL)
- Dilute 1uL stock gDNA with 99uL RNAse-free H20
- 2% Agarose Gel
Overview[edit]
- Cell Preparation
- Bisulfite Conversion
- Elution
- PCR amplification
- Agarose Gel Electrophoresis
Procedures[edit]
- Turn on the incubator heat it up to 50C
- Cell Preparation
Resuspended cells in T25 flask by repeat pipetting
Perform cell counting
- Cell Count of GM20431 P20: 207,656 cells/mL
To get 1,000,000 cells: 1,000,000/(207,656cells/mL)=4.816mL
aspirate 4mL + 816uL cell suspension and transfer to a 50mL centrifuge tube
Spin down at 10,000 rpm for 5 mins
Aspirate supernatant completely
Resuspended cells with 1000uL UV treated PBS (1000cells/uL)
- Cell Dilution
Performed cell dilution to reach concentration of 11.11 cells/uL and 1.11 cells/uL
- 111.11 cells/uL
Dilute 10uL (1000cells/uL)cell suspension with 80uL UV TREATED PBS IN PCR TUBE
- 11.11 cells/uL
Dilute 10uL (111.11 cells/uL) cell suspension with 90 uL UV TREATED PBS IN PCR TUBE
- 1.11 cells/uL
Dilute 10uL (11.11 cells/uL) cell suspension with 90 uL UV TREATED PBS IN PCR TUBE
- Sample Digestion with Proteinase K
1rxn x 6 -------------------------------------------- RNAse-free H2O 0.0 0.0 Protinase K 1.0 1.0 M-Digestion Buffer (2X) 10.0 10.0 1.11 cells/uL 9.0 -------------------------------------------- 20uL 20uL
Vortex and spindown
Incubate the samples at 50C for 20 mins
- Bisulfite Conversion of DNA
Add in 130uL of CT conversion Reagent Solution directly to the digested samples
Vortex and spin down
Perform reaction in thermocycler
Step1 98C, 8m Step2 64C, 3.5hr Step4 4C, storage for up to 20 hr
Add 600uL of M-Bindin Buffer into a IC Column
Load 150uL of samples into IC column
CLOSE CAP AND MIX BY INVERTING THE COLUMN SEVERAL TIMES
Centrifuge at 20,000g for 30s Discard flow through
Add 100uL M-Wash Buffer to column Repeat Centrifuge
Add 200uL of M-Desulphonation Buffer to column let stand at RT for 20m Repeat centrifuge step
Add 200uL of M-Wash Buffer to the column Repeat Centrifuge [REPEAT WASHING STIP FOR ONE SET ONLY]
Place column in a 1.5mL tube Add in 8uL of M-Elution Buffer directly to the column matix Repeat Centrifuge
- PCR
Sample A 10 cell GM20431 (Single Washing) A B C ------------------------------------------------- CHR22 CHR21 CHR8 2X iQ Super Mix 20 20 20 Primer F (3.3uM) 6 6 6 Primer R (3.3uM) 6 6 6 gDNA 8 8 8 ------------------------------------------------- Total 40uL
Sample B 10 cell GM20431 (Double Washing) A B C ------------------------------------------------- CHR22 CHR21 CHR8 2X iQ Super Mix 20 20 20 Primer F (3.3uM) 6 6 6 Primer R (3.3uM) 6 6 6 gDNA 8 8 8 ------------------------------------------------- Total 40uL
A-CHR22 F/R
B-CHR21 F/R
C-CHR8 F/R
Perform PCR reaction in thermocycler
Step1 96C, 3m Step2 95C, 30s Step3 62C, 1m Step4 72C, 1m Step5 Go to step2 repeat 39 times Step6 72C, 5m Step7 4C, Forever
- Gel Electrophoresis
Gel 1 Well 1 2 3 4 5 6 7 8 ----------------------------------------------------------------- Content Blank AA AB AC BA BB BC Ladder Sample 0 10 10 10 10 10 10 3 6X Loading Dye 0 2 2 2 2 2 2 3 ----------------------------------------------------------------- Total 14uL 9uL
- Run gel at 135 V for 20 min.
Results[edit]
File:ZhangLab 2 2009-03-26 11hr 16min crop.jpg
- All 3 pairs of primers showed up for 1 washing before elution
- Only CHR21 primer showed up for double washing before elution
Suggestion[edit]
- Repeat experiment with single cell
- Only perform one washing