Dinh:Protocols/Capturing Nov13: Difference between revisions
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>Dinh |
>Alan6017518 |
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!Reagent | !Reagent | ||
!Final Conc. | !Final Conc. | ||
! | !#1 (3x) | ||
!#2 | |||
!#3 | |||
|- | |- | ||
|ddH20 | |ddH20 | ||
| | | | ||
|10ul | |10ul | ||
|15ul | |||
|19ul | |||
|- | |- | ||
|Jurkat gDNA (203.9ng/ul) | |Jurkat gDNA (203.9ng/ul) | ||
| | | varies per tube | ||
|10ul | |10ul | ||
|5ul | |||
|1ul | |||
|- | |- | ||
|Conversion Reagent (prepared 11/6) | |Conversion Reagent (prepared 11/6) | ||
| | | | ||
|130ul | |||
|130ul | |||
|130ul | |130ul | ||
|} | |} | ||
Line 36: | Line 44: | ||
9. Place the column into 1.5ml microcentrifuge tube. Add 12ul M-Elution Buffer into center of column matrix. Wait 1 min. Centrifuge at 15K rpm for 30 s. | 9. Place the column into 1.5ml microcentrifuge tube. Add 12ul M-Elution Buffer into center of column matrix. Wait 1 min. Centrifuge at 15K rpm for 30 s. | ||
10. Measure the DNA with Nanodrop. | 10. Measure the DNA with Nanodrop. | ||
{| border = "1" | |||
!Sample # | |||
!Input Jurkat gDNA (ng) | |||
!Output converted gDNA (ng) | |||
! % yield | |||
|- | |||
| 1 | |||
| 2039ng | |||
| 198.8ng/ulx10ul = 1988ng | |||
| 97% | |||
|- | |||
| 2 | |||
| 1019.5ng | |||
| 1421.7ng | |||
| - | |||
| 3 | |||
| 203.9ng | |||
| 192ng | |||
| | |||
=B. November 23, 2009 - PCR Amplification of Converted DNA= | =B. November 23, 2009 - PCR Amplification of Converted DNA= |
Revision as of 20:05, 25 November 2009
A. November 13, 2009 Bisulfite conversion of DNA using Zymo EZ DNA Methylation-Gold Kit
Reaction Mix (1x, 150ul):
Reagent | Final Conc. | #1 (3x) | #2 | #3 |
---|---|---|---|---|
ddH20 | 10ul | 15ul | 19ul | |
Jurkat gDNA (203.9ng/ul) | varies per tube | 10ul | 5ul | 1ul |
Conversion Reagent (prepared 11/6) | 130ul | 130ul | 130ul |
Reaction Program:
98C -> 10 min 64C -> 2.5 hours 4C -> overnight
Column Purification *Washing was done twice:
1. Place a Zymo-Spin IC Column into a provided Collection tube. 2. Add 600 ul of M-Binding Buffer to spin column 3. Load the reaction mix into the spin column. 4. Close cap and mix by inverting 10 times. Centrifuge at 15K rpm for 30 s. Discard flow through. 5. Add 100ul of M-Wash Buffer. Centrifuge at 15K rpm for 30 s. Discard flow through. 6. Add 200ul M-Desulphonation Buffer and wait for 20 min. Centrifuge at 15K rpm for 30 s. Discard flow through. 7. Add 200ul M-Wash Buffer. Centrifuge at 15K rpm for 30 s. Discard flow through. 8. Add another 200ul M-Wash Buffer. Centrifuge at 15K rpm for 30 s. Discard flow through. 9. Place the column into 1.5ml microcentrifuge tube. Add 12ul M-Elution Buffer into center of column matrix. Wait 1 min. Centrifuge at 15K rpm for 30 s. 10. Measure the DNA with Nanodrop.
Sample # | Input Jurkat gDNA (ng) | Output converted gDNA (ng) | % yield | |||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
1 | 2039ng | 198.8ng/ulx10ul = 1988ng | 97% | |||||||||||||||||||||||||
2 | 1019.5ng | 1421.7ng | - | 3 | 203.9ng | 192ng |
B. November 23, 2009 - PCR Amplification of Converted DNA
-------------------------------------------------- 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 -------------------------------------------------- Reaction mix:
A. Jurkat gDNA - 203.9 ng/ul B. converted gDNA by Alan (200ng/ul) C. 11-13 converted gDNA (400ng/ul) D. 11-13 converted gDNA (135ng/ul) Program: 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 Step8 END C. Capturing Protocol CpG30KPositive control: Jurkat converted DNA Negative control: 1ul RNAse free ddH20 |