AlanFung:LabNotes/EZ/2009-3-12
EZ DNA Methylation Direct-GM20431 1,000 cells with Jurkat gDNA [edit]
Objective[edit]
- Confirmation of the bisulfite conversion of GM20431 for 1,000 cells
- Using same amount of Jurkat gDNA from 1,000 cells to test the bisulfite conversion
- Increase low mass ladder volume to 3uL for a better image
Samples & Materials[edit]
- GM20431 cells P15
- 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
- Agarose Gel
Overview[edit]
- Cell Preparation
- Bisulfite Conversion
- PCR amplification
- Agarose Gel Electrophoresis
Procedures[edit]
- Cell Preparation
Resuspended cells in T25 flask by repeat pipetting Perform cell counting
- Cell Countof GM20431 P15: 346,430 cells/mL
To get 1,000,000 cells: 1,000,000/(346,430/mL)=2.886mL aspirate 2mL + 886uL cell suspension and transfer to a 50mL centrifuge tube Spin down at 10,000rpm for 3 mins Aspirate supernatant completely Resuspended cells with 1000uL UV treated PBS (1000cells/uL)
- Cell Dilution
Performed cell dilution to reach concentration of 111.11 cells/uL Dilute 8uL (1000cells/uL)cell suspension with 800uL UV treated PBS
- Sample Digestion with Proteinase K
1rxn x 2 ------------------------------------ RNAse-free H2O 0.0 Protinase K 1.0 M-Digestion Buffer (2X) 10.0 ------------------------------------ Mix by repeat pipetting Cells 9.0 ------------------------------------ 20uL
Incubate the samples at 50C for 20 mins
- Bisulfite Conversion of DNA
Add in 130uL of CT conversion Reagent Solution into a labeled PCR tube Extract 20uL of sample supernatant Mix by repeat pipetting Centrifuge briefly to ensure no droplets are in the cap or side of the tube
Perform reaction in thermocycler
Step1 98C, 8m Step2 64C, 3.5hr Step3 62C, 1m Step4 4C, storage for up to 20 hr
Add 600uL of M-Bindin Buffer into a IC Column and place column into a collection tube Load samples into IC column Close cap and mix by inverting the column several times Centrifuge at (max speed) 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 step
Place column in a 1.5mL tube Add in 10uL of M-Elution Buffer directly to the column matix Repeat Centrifuge
- PCR
Sample A 1,000cell GM20431 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 0.5 0.5 0.5 RNAse free H20 7.5 7.5 7.5 ------------------------------------------------- Total 40uL
Sample B 1,000cell GM20431 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 0.5 0.5 0.5 RNAse free H20 7.5 7.5 7.5 ------------------------------------------------- Total 40uL
Sample C 6ng Jurkat gDNA 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 0.5 0.5 0.5 RNAse free H20 7.5 7.5 7.5 ------------------------------------------------- Total 40uL
Sample D 6ng Jurkat gDNA 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 0.5 0.5 0.5 RNAse free H20 7.5 7.5 7.5 ------------------------------------------------- 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
- 1st and 2nd Gel Electrophoresis
Gel 1 Well 1 2 3 4 5 6 7 8 ----------------------------------------------------------------- Content Blank AA AB AC CA CB CC Ladder Sample 0 6 6 6 6 6 6 3 0.5X TBE Buffer 0 4 4 4 4 4 4 3 6X Loading Dye 0 4 4 4 4 4 4 3 ----------------------------------------------------------------- Total 14uL 9uL
Gel 2 Well 1 2 3 4 5 6 7 8 ----------------------------------------------------------------- Content Blank BA BB BC DA DB DC Ladder Sample 0 6 6 6 6 6 6 3 0.5X TBE Buffer 0 4 4 4 4 4 4 3 6X Loading Dye 0 4 4 4 4 4 4 3 ----------------------------------------------------------------- Total 14uL 9uL
- Run both gels at the same time at 135 V for 20 min.
Results[edit]
File:ZhangLab 2 2009-03-12 15hr 26min crop.jpg
*6 Bands show up for each gel around 200bp for 3 different pairs of primer *Gel 1 Jurkat gDNA CT conversion PCR with primer CHR8's signal is much weaker than the ones in gel 2. Probably due to dropping of the column C during the centrifuge step
Suggestion[edit]
- Lower cell number to 100 and 10 cells, using live cells.