Jie:LabNotes/CpgSeq/2008-10-10: Difference between revisions
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PGPF1 57.8ng/ul x 50ul. 260/280=0.96 | PGPF1 57.8ng/ul x 50ul. 260/280=0.96 | ||
==Bisulfite conversion of DNA using Zymo EZ DNA Methylation-Gold kit == | |||
==Bisulfite conversion of DNA using Zymo EZ DNA Methylation-Gold kit (4th) == | |||
{| border="1" cellpadding="5" cellspacing="0" align="center" | {| border="1" cellpadding="5" cellspacing="0" align="center" | ||
Line 21: | Line 22: | ||
| align="center" style="background:#f0f0f0;"|'''260:280/260:230''' | | align="center" style="background:#f0f0f0;"|'''260:280/260:230''' | ||
|- | |- | ||
|2||BJ_iPS_11||140ng/ul x 2 tubes||14.3ul||5.7ul||130ul|| | |2||BJ_iPS_11||140ng/ul x 2 tubes||14.3ul||5.7ul||130ul||101.9ng/ul x 20ul||1.6/0.9 | ||
|- | |- | ||
|16||IMB90_2||50ng/ul||20ul||0ul||130ul|| | |16||IMB90_2||50ng/ul x 2 tubes||20ul||0ul||130ul||68ng/ul x 10ul||1.48/0.93 | ||
|- | |- | ||
|17||PGPF1||57.8ng/ul||20ul||0ul||130ul|| | |17||PGPF1||57.8ng/ul x 3 tubes||20ul||0ul||130ul||119.5ng/ul x 30ul||1.79/1.28 | ||
|} | |} | ||
Line 40: | Line 41: | ||
*Measure the DNA with Nanodrop: | *Measure the DNA with Nanodrop: | ||
RNA | RNA | ||
==set up the capture system== | ==set up the capture system== | ||
Line 50: | Line 52: | ||
| align="center" style="background:#f0f0f0;"|'''10xLigase buffer''' | | align="center" style="background:#f0f0f0;"|'''10xLigase buffer''' | ||
| align="center" style="background:#f0f0f0;"|'''template+cpg30k(50.7ng/ul)+suppressor(8nM each)vol+H2O''' | | align="center" style="background:#f0f0f0;"|'''template+cpg30k(50.7ng/ul)+suppressor(8nM each)vol+H2O''' | ||
| align="center" style="background:#f0f0f0;"|'''template+v2.1(21ng/ul) vol+H2O''' | | align="center" style="background:#f0f0f0;"|'''template+v2.1(21ng/ul;7/23/08) vol+H2O''' | ||
| align="center" style="background:#f0f0f0;"|'''template+v2.2( | | align="center" style="background:#f0f0f0;"|'''template+v2.2(24ng/ul;7/7/08) vol+H2O''' | ||
| align="center" style="background:#f0f0f0;"|'''template+v2.3( | | align="center" style="background:#f0f0f0;"|'''template+v2.3(28ng/ul;7/7/08) vol+H2O''' | ||
|- | |- | ||
|2||BJ_iPS_11|| | |2||BJ_iPS_11||101.9ng/ul x 20ul||1ul||4+1+1+5ul||4+1.5+4.5||8+1.5+2.5||4+1.5+3.5 | ||
|- | |- | ||
|16|| | |16||IMB90_2||68ng/ul x 20ul||1ul||4+1+1+5ul||4+1.5+4.5||8+1.5+2.5||4+1.5+3.5 | ||
|- | |- | ||
|17|| | |17||PGPF1||119.5ng/ul x 30ul||1ul||4+1+1+5ul||4+1.5+4.5||8+1.5+2.5||4+1.5+3.5 | ||
| | | |- | ||
| ||bis_Jurkat||200ng/ul||1ul||1+1+1+6ul|| | |||
|- | |||
| ||Jurkat||200ng/ul||1ul||1+ cosmic probe 2ul+h2O 6ul|| | |||
|- | |- | ||
| ||negative control||0||1ul||1+1+1+6ul||1+1.5+6.5||2+1.5+5.5||1.5+1.5+6 | | ||negative control||0||1ul||1+1+1+6ul||1+1.5+6.5||2+1.5+5.5||1.5+1.5+6 | ||
Line 71: | Line 76: | ||
95c 10min -> 55C 16h ->add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2mM dNTP) -> 55C 4h -> <BR>9 cycels (95C 1min -> 55C for 4h) -> 95C 1min -> 37C 1min -> <BR>add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> 4C hold. | 95c 10min -> 55C 16h ->add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2mM dNTP) -> 55C 4h -> <BR>9 cycels (95C 1min -> 55C for 4h) -> 95C 1min -> 37C 1min -> <BR>add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> 4C hold. | ||
==PCR(10/13/2008)== | |||
x8 | |||
Template 10ul | |||
2X iProof Mastermix 50ul 400ul | |||
AmpF6.2SoL (10uM) 4ul 32ul | |||
AmpR6.2SoL (10uM) 4ul 32ul | |||
50X SYBG I 0.8ul 6.4ul | |||
H2O 31.2ul 249.6ul | |||
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 10-> 72C 5 min -> 15C hold. | |||
[[Image:20081013_captured_PCR_No_2_16.jpg]]20081013_captured_PCR_No_2_16 | |||
[[Image:20081013_captured_PCR_No_17.jpg]]20081013_captured_PCR_No_17 | |||
==Digestion of IMR_90 and PGP1F with Rnase I (10/13/08)== | |||
PGP1F(35ng/ul) 200ul + 10U/ul Rnase I 8ul -> 37C 30mins -> 70C 20mins. | |||
IMR90(25ng/ul) 200ul + 10U/ul Rnase I 8ul -> 37C 30mins -> 70C 20min. | |||
Purify with Microcon column. | |||
Yield: | |||
IMR90 39.7ng/ul x 40ul. 260/280=1.76, 260/230=0.8 | |||
PGPF1 72.9ng/ul x 50ul. 260/280=1.82, 260/230=1.07 | |||
==Bisulfite conversion of DNA using Zymo EZ DNA Methylation-Gold kit == | |||
{| border="1" cellpadding="5" cellspacing="0" align="center" | |||
|- | |||
| align="center" style="background:#f0f0f0;"|''' ''' | |||
| align="center" style="background:#f0f0f0;"|'''sample ''' | |||
|align="center" style="background:#f0f0f0;"|'''sample concentration''' | |||
| align="center" style="background:#f0f0f0;"|'''sample volumn''' | |||
| align="center" style="background:#f0f0f0;"|'''ddH2O ''' | |||
| align="center" style="background:#f0f0f0;"|'''conversion reagents''' | |||
| align="center" style="background:#f0f0f0;"|'''conversed DNA concentration and volumn''' | |||
| align="center" style="background:#f0f0f0;"|'''260:280/260:230''' | |||
|- | |||
|2||BJ_iPS_11||140ng/ul x 1 tubes||14.3ul||5.7ul||130ul||147.4ng/ul x 10ul||1.6/0.9 | |||
|- | |||
|16||IMB90_2||39.7ng/ul x 2 tubes||20ul||0ul||130ul||66ng/ul x 10ul||1.48/0.93 | |||
|- | |||
|17||PGPF1||72.9ng/ul x 2 tubes||20ul||0ul||130ul||194.1ng/ul x 10ul||1.79/1.28 | |||
|- | |||
|Jurkat||||200ng/ul x 2 tubes||10ul||10ul||130ul||309.8ng/ul x 10ul | |||
|} | |||
*Add 900ul ddH2O, 300ul M-Dilution Buffer and 50ul M-Dissolving Buffer to a tube of CT Conversion Reagent. | |||
*Add 130ul of CT conversion reagent to samples. | |||
*98C 10min -> 64C 150min -> 4c overnight. | |||
*Add 600ul M-Binding buffer to spin columns. | |||
*Add the converted samples to the columns, close the gap and mix by inverting several times. | |||
*Spin at 15,000rpm for 30sec. | |||
*Add 100ul M-Wash Buffer, spin for 30 sec, discard flow-through. | |||
*Add 200ul M-Desulphonation Buffer, wait for 20min, spin at 15,000 rpm for 30sec. | |||
*Add 200ul M-Wash Buffer, spin for 30 sec. | |||
*Place the columns into 1.5ml tubes, add 10ul M-Elution Buffer. Wait for 1min, spin at 15,000rpm for 30sec. | |||
*Measure the DNA with Nanodrop: | |||
RNA | |||
==set up the capture system== | |||
{| border="1" cellpadding="5" cellspacing="0" align="center" | |||
|- | |||
| align="center" style="background:#f0f0f0;"|''' ''' | |||
| align="center" style="background:#f0f0f0;"|'''sample ''' | |||
|align="center" style="background:#f0f0f0;"|'''sample concentration''' | |||
| align="center" style="background:#f0f0f0;"|'''10xLigase buffer''' | |||
| align="center" style="background:#f0f0f0;"|'''template+cpg30k(50.7ng/ul)+suppressor(8nM each)vol+H2O''' | |||
| align="center" style="background:#f0f0f0;"|'''template+v2.1(21ng/ul;7/23/08) vol+H2O''' | |||
| align="center" style="background:#f0f0f0;"|'''template+v2.2(21ng/ul;7/23/08) vol+H2O''' | |||
| align="center" style="background:#f0f0f0;"|'''template+v2.3(28ng/ul;7/7/08) vol+H2O''' | |||
|- | |||
|2||BJ_iPS_11||147.4ng/ul x 10ul||1ul||||||10+1.5|| | |||
|- | |||
|16||IMB90_2||66ng/ul x 10ul||1ul||||||10+1.5|| | |||
|- | |||
|17||PGPF1||191.4ng/ul x 10ul||1ul||||||10+1.5+2.5|| | |||
|- | |||
| ||bis_Jurkat||175ng/ul||1ul||||||1+1.5+6.5ul|| | |||
|- | |||
| ||bis_Jurkat||175ng/ul||1ul||||||1+1.5(7/7/08)+6.5|| | |||
|- | |||
| ||negative control||0||1ul||||||0+1.5+7.5|| | |||
|} | |||
SLN mix is prepared as the above. | |||
==PCR(10/16/2008)== | |||
x3 | |||
Template 10ul | |||
2X iProof Mastermix 50ul 150ul | |||
AmpF6.2SoL (10uM) 4ul 12ul | |||
AmpR6.2SoL (10uM) 4ul 12ul | |||
50X SYBG I 0.8ul 2.4ul | |||
H2O 31.2ul 93.6ul | |||
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 10-> 72C 5 min -> 15C hold. | |||
[[Image:20081016_captured_PCR_No_2_16_17.jpg]]20081016_captured_PCR_No_2_16_17 | |||
yield: No_2: | |||
cpg30k + suppressor: 34ng/ul x 15ul | |||
V2.1: 23ng/ul x 15ul | |||
V2.3: 13ng/ul x 15ul | |||
mix 2ul cpg30K + 3ul V2.1 + 5ul V2.3 = 6ng/ul x 10ul | |||
No_16: | |||
cpg30k + suppressor: 17ng/ul x 15ul | |||
V2.1: 13ng/ul x 15ul | |||
V2.3: 11ng/ul x 15ul | |||
mix 2.5ul cpg30k + 3ul V2.1 + 3ul V2.3 = 3ng/ul x 8.5ul | |||
No_17: | |||
cpg30k + suppressor: 16ng/ul x 15ul | |||
V2.1: 18ng/ul x 15ul | |||
V2.3: 11ng/ul x 15ul | |||
mix 3ul cpg30K + 3ul V2.1 + 4ul V2.3 = 5ng/ul x 10ul |
Latest revision as of 22:11, 8 January 2009
Digestion of PGP1F with Rnase I[edit]
PGP1F(35ng/ul) 100ul + 10U/ul Rnase I 2ul -> 37C 30mins -> 70C 20mins. IMR90(25ng/ul) 100ul + 10U/ul Rnase I 2ul -> 37C 30mins -> 70C 20min. Purify with Microcon column. Yield: IMR90 50ng/ul x 40ul. 260/280=0.96 PGPF1 57.8ng/ul x 50ul. 260/280=0.96
Bisulfite conversion of DNA using Zymo EZ DNA Methylation-Gold kit (4th)[edit]
sample | sample concentration | sample volumn | ddH2O | conversion reagents | conversed DNA concentration and volumn | 260:280/260:230 | |
2 | BJ_iPS_11 | 140ng/ul x 2 tubes | 14.3ul | 5.7ul | 130ul | 101.9ng/ul x 20ul | 1.6/0.9 |
16 | IMB90_2 | 50ng/ul x 2 tubes | 20ul | 0ul | 130ul | 68ng/ul x 10ul | 1.48/0.93 |
17 | PGPF1 | 57.8ng/ul x 3 tubes | 20ul | 0ul | 130ul | 119.5ng/ul x 30ul | 1.79/1.28 |
- Add 900ul ddH2O, 300ul M-Dilution Buffer and 50ul M-Dissolving Buffer to a tube of CT Conversion Reagent.
- Add 130ul of CT conversion reagent to samples.
- 98C 10min -> 64C 150min -> 4c overnight.
- Add 600ul M-Binding buffer to spin columns.
- Add the converted samples to the columns, close the gap and mix by inverting several times.
- Spin at 15,000rpm for 30sec.
- Add 100ul M-Wash Buffer, spin for 30 sec, discard flow-through.
- Add 200ul M-Desulphonation Buffer, wait for 20min, spin at 15,000 rpm for 30sec.
- Add 200ul M-Wash Buffer, spin for 30 sec.
- Place the columns into 1.5ml tubes, add 10ul M-Elution Buffer. Wait for 1min, spin at 15,000rpm for 30sec.
- Measure the DNA with Nanodrop:
RNA
set up the capture system[edit]
sample | sample concentration | 10xLigase buffer | template+cpg30k(50.7ng/ul)+suppressor(8nM each)vol+H2O | template+v2.1(21ng/ul;7/23/08) vol+H2O | template+v2.2(24ng/ul;7/7/08) vol+H2O | template+v2.3(28ng/ul;7/7/08) vol+H2O | |
2 | BJ_iPS_11 | 101.9ng/ul x 20ul | 1ul | 4+1+1+5ul | 4+1.5+4.5 | 8+1.5+2.5 | 4+1.5+3.5 |
16 | IMB90_2 | 68ng/ul x 20ul | 1ul | 4+1+1+5ul | 4+1.5+4.5 | 8+1.5+2.5 | 4+1.5+3.5 |
17 | PGPF1 | 119.5ng/ul x 30ul | 1ul | 4+1+1+5ul | 4+1.5+4.5 | 8+1.5+2.5 | 4+1.5+3.5 |
bis_Jurkat | 200ng/ul | 1ul | 1+1+1+6ul | ||||
Jurkat | 200ng/ul | 1ul | 1+ cosmic probe 2ul+h2O 6ul | ||||
negative control | 0 | 1ul | 1+1+1+6ul | 1+1.5+6.5 | 2+1.5+5.5 | 1.5+1.5+6 |
dNTP concentration 10mM(NEB) ddH2O Ligase Buffer Ligase Amplitaq dNTP 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2mM dNTP: 0.58ul 0.1ul 0.1ul 0.2ul (10mM)0.02ul x300 174ul 30ul 30ul 60ul 6ul
95c 10min -> 55C 16h ->add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2mM dNTP) -> 55C 4h ->
9 cycels (95C 1min -> 55C for 4h) -> 95C 1min -> 37C 1min ->
add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> 4C hold.
PCR(10/13/2008)[edit]
x8 Template 10ul 2X iProof Mastermix 50ul 400ul AmpF6.2SoL (10uM) 4ul 32ul AmpR6.2SoL (10uM) 4ul 32ul 50X SYBG I 0.8ul 6.4ul H2O 31.2ul 249.6ul
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 10-> 72C 5 min -> 15C hold.
File:20081013 captured PCR No 2 16.jpg20081013_captured_PCR_No_2_16
File:20081013 captured PCR No 17.jpg20081013_captured_PCR_No_17
Digestion of IMR_90 and PGP1F with Rnase I (10/13/08)[edit]
PGP1F(35ng/ul) 200ul + 10U/ul Rnase I 8ul -> 37C 30mins -> 70C 20mins. IMR90(25ng/ul) 200ul + 10U/ul Rnase I 8ul -> 37C 30mins -> 70C 20min. Purify with Microcon column. Yield: IMR90 39.7ng/ul x 40ul. 260/280=1.76, 260/230=0.8 PGPF1 72.9ng/ul x 50ul. 260/280=1.82, 260/230=1.07
Bisulfite conversion of DNA using Zymo EZ DNA Methylation-Gold kit[edit]
sample | sample concentration | sample volumn | ddH2O | conversion reagents | conversed DNA concentration and volumn | 260:280/260:230 | |
2 | BJ_iPS_11 | 140ng/ul x 1 tubes | 14.3ul | 5.7ul | 130ul | 147.4ng/ul x 10ul | 1.6/0.9 |
16 | IMB90_2 | 39.7ng/ul x 2 tubes | 20ul | 0ul | 130ul | 66ng/ul x 10ul | 1.48/0.93 |
17 | PGPF1 | 72.9ng/ul x 2 tubes | 20ul | 0ul | 130ul | 194.1ng/ul x 10ul | 1.79/1.28 |
Jurkat | 200ng/ul x 2 tubes | 10ul | 10ul | 130ul | 309.8ng/ul x 10ul |
- Add 900ul ddH2O, 300ul M-Dilution Buffer and 50ul M-Dissolving Buffer to a tube of CT Conversion Reagent.
- Add 130ul of CT conversion reagent to samples.
- 98C 10min -> 64C 150min -> 4c overnight.
- Add 600ul M-Binding buffer to spin columns.
- Add the converted samples to the columns, close the gap and mix by inverting several times.
- Spin at 15,000rpm for 30sec.
- Add 100ul M-Wash Buffer, spin for 30 sec, discard flow-through.
- Add 200ul M-Desulphonation Buffer, wait for 20min, spin at 15,000 rpm for 30sec.
- Add 200ul M-Wash Buffer, spin for 30 sec.
- Place the columns into 1.5ml tubes, add 10ul M-Elution Buffer. Wait for 1min, spin at 15,000rpm for 30sec.
- Measure the DNA with Nanodrop:
RNA
set up the capture system[edit]
sample | sample concentration | 10xLigase buffer | template+cpg30k(50.7ng/ul)+suppressor(8nM each)vol+H2O | template+v2.1(21ng/ul;7/23/08) vol+H2O | template+v2.2(21ng/ul;7/23/08) vol+H2O | template+v2.3(28ng/ul;7/7/08) vol+H2O | |
2 | BJ_iPS_11 | 147.4ng/ul x 10ul | 1ul | 10+1.5 | |||
16 | IMB90_2 | 66ng/ul x 10ul | 1ul | 10+1.5 | |||
17 | PGPF1 | 191.4ng/ul x 10ul | 1ul | 10+1.5+2.5 | |||
bis_Jurkat | 175ng/ul | 1ul | 1+1.5+6.5ul | ||||
bis_Jurkat | 175ng/ul | 1ul | 1+1.5(7/7/08)+6.5 | ||||
negative control | 0 | 1ul | 0+1.5+7.5 |
SLN mix is prepared as the above.
PCR(10/16/2008)[edit]
x3 Template 10ul 2X iProof Mastermix 50ul 150ul AmpF6.2SoL (10uM) 4ul 12ul AmpR6.2SoL (10uM) 4ul 12ul 50X SYBG I 0.8ul 2.4ul H2O 31.2ul 93.6ul
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 10-> 72C 5 min -> 15C hold.
File:20081016 captured PCR No 2 16 17.jpg20081016_captured_PCR_No_2_16_17
yield: No_2:
cpg30k + suppressor: 34ng/ul x 15ul V2.1: 23ng/ul x 15ul V2.3: 13ng/ul x 15ul mix 2ul cpg30K + 3ul V2.1 + 5ul V2.3 = 6ng/ul x 10ul
No_16: cpg30k + suppressor: 17ng/ul x 15ul V2.1: 13ng/ul x 15ul V2.3: 11ng/ul x 15ul mix 2.5ul cpg30k + 3ul V2.1 + 3ul V2.3 = 3ng/ul x 8.5ul
No_17: cpg30k + suppressor: 16ng/ul x 15ul V2.1: 18ng/ul x 15ul V2.3: 11ng/ul x 15ul mix 3ul cpg30K + 3ul V2.1 + 4ul V2.3 = 5ng/ul x 10ul