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=A. November 13, 2009 Bisulfite conversion of DNA using Zymo EZ DNA Methylation-Gold Kit= Reaction Mix (1x, 150ul): {| border="1" !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 (use M-Elution Buffer to blank) {| border = "1" !Sample # !Input Jurkat gDNA (ng) !Output converted gDNA (ng) ! % yield |- | 1 | 2039ng | 198.8ng/ulx10ul = 1988ng | 97% |- | 2 | 1019.5ng | 79ng/ulx10.5ul = 829.5ng | 81% |- | 3 | 203.9ng | 20ng/ulx9.6ul = 192ng | 94% |} Notes: Next time, repeat using 2x of sample 1 reaction and use 14ul M-Elution buffer to elute first column, then use flow-through to elute second column. =B. November 23, 2009 - PCR Amplification of Converted DNA= *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 -------------------------------------------------- Reaction mix: {| border="1" !Reagent !Final Conc. !Vol (1x) |- |ddH20 | |7.5ul |- | NEB Tag 2x Master Mix |1x |20ul |- |Forward Primer (Chr8/21/22) (3.3uM) |~0.5uM |6ul |- |Reverse Primer (Chr8/21/22) (3.3uM) |~0.5uM |6ul |- |A/B/C/D | |0.5ul |- !Total | |40ul |} 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 ==Results== [[Image:Dinh 2009-11-25 Bisulfite Converted Jurket gDNA verification.jpg]] =C. November 25, 2009 - Capturing Protocol CpG30K= Note: Used CpG30K (11/11, 7ng/ul) 100x probes to template ratio Program: CAP30K 95 C -> 10 min 55 C -> forever (*) 55 C -> 4 hr 95 C -> 1 min RAMP: 0.1 C/s -> 55 C 55 C -> 4 hr Goto 4, 8x 55 C -> forever 95 C -> 5 min 37 C -> 1 hr (**) 94 C -> 5 min 4 C -> forever * after at least 16 hr, add 1 ul SLN mix per reaction ** Add 2 ul Lambda Exonuclease I/III mix as soon as temperature is lowered to 37C ==Nov 26 2009== Added 1ul SLN Mix {| {{table}} border = "1" | align="center" style="background:#f0f0f0;"|'''Reagent''' | align="center" style="background:#f0f0f0;"|'''Stock Conc''' | align="center" style="background:#f0f0f0;"|'''Final Conc''' | align="center" style="background:#f0f0f0;"|'''Volume to Add(ul)''' |- | Stofell(U/uL)||10||2||0.8 |- | Ampligase(U/ul)||5||0.5||0.4 |- | dNTP(mM)||1||0.2||0.8 |- | 10xLigase||10||1||0.4 |- | H20|| - || - || 1.6 |- | || || || 4 TOTAL |- |} ==Nov 29 2009== Added 2ul ExoI/III mix to each rxn. =Dec 2 2009 PCR Amplification of Capture Products= PCR Amplification {| {table} border ="1" | align="center" style="background:#f0f0f0;"|'''Reagent''' | align="center" style="background:#f0f0f0;"|'''Vol (1x)''' | align="center" style="background:#f0f0f0;"|'''Vol (5x)''' |- | Template (*) || 10ul || |- |2x iProof MM || 50ul || 250ul |- |AmpF6.2Sol (10uM) || 4ul || 20ul |- |AmpR6.2Sol (10uM) || 4ul || 20ul |- |50x SYBR Green || 0.8ul || 4ul |- | RNase Free H20 || 31.2ul || 156ul |- | Total || 100ul || 450ul |- |} (*) Templates: A. 300ng/ul Template B. 153ng/ul Template C. 79ng/ul Template D. Negative capture control E. RNAse free H20 Program 98C -> 30s 98C -> 10s 58C -> 20s 72C -> 20s Plate Read Go to 2 for 7x 98C -> 10s 72C -> 20s Plate Read Go to 7 for 10x 72C -> 3min 15C -> forever End PAGE Quantification [[Image:Dinh Capture reaction after PCR amp.jpg]]
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