Dinh:Protocols/Capturing Nov13: Difference between revisions

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A. Bisulfite conversion of DNA using Zymo EZ DNA Methylation-Gold Kit
=A. November 13, 2009 Bisulfite conversion of DNA using Zymo EZ DNA Methylation-Gold Kit=


Reaction Mix (1x, 150ul):  
Reaction Mix (1x, 150ul):  
  Jurkat DNA (203.9ng/ul) - 10ul  
{| border="1"
  RNase free ddH20 - 10 ul
!Reagent
  (DNA = 2.039ug)
!Final Conc.
  Conversion Reagent (prepared 11/6) - 130 ul
!#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:
Reaction Program:
Line 12: Line 33:
   4C -> overnight
   4C -> overnight


Place a Zymo-Spin IC Column into a provided Collection tube.
Column Purification *Washing was done twice:
Add 600 ul of M-Binding Buffer to spin column
1. Place a Zymo-Spin IC Column into a provided Collection tube.
Load the reaction mix into the spin column.
2. Add 600 ul of M-Binding Buffer to spin column
Close cap and mix by inverting 10 times.
3. Load the reaction mix into the spin column.
Centrifuge at 15K rpm for 30 s. Discard flow through.
4. Close cap and mix by inverting 10 times. Centrifuge at 15K rpm for 30 s. Discard flow through.
Add 100ul of M-Wash Buffer.
5. Add 100ul of M-Wash Buffer. Centrifuge at 15K rpm for 30 s. Discard flow through.
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.
Add 200ul M-Desulphonation Buffer and wait for 20 min.
7. Add 200ul M-Wash Buffer. Centrifuge at 15K rpm for 30 s. Discard flow through.
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.
Add 200ul M-Wash Buffer.
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.
Centrifuge at 15K rpm for 30 s. Discard flow through.
10. Measure the DNA with Nanodrop (use M-Elution Buffer to blank)
Add another 200ul M-Wash Buffer.
Centrifuge at 15K rpm for 30 s. Discard flow through.


Place the column into 1.5ml microcentrifuge tube. Add 12ul M-Elution Buffer into center of column matrix. Wait 1 min.  
{| border = "1"
Centrifuge at 15K rpm for 30 s. Discard flow through.
!Sample #
Measure the DNA with Nanodrop.
!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%
|}


B. Capturing Protocol CpG30K
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.


Positive control: Jurkat converted DNA
=B. November 23, 2009 - PCR Amplification of Converted DNA=
Negative control: 1ul RNAse free ddH20
*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]]

Latest revision as of 21:46, 7 January 2010

A. November 13, 2009 Bisulfite conversion of DNA using Zymo EZ DNA Methylation-Gold Kit[edit]

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 (use M-Elution Buffer to blank)
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[edit]

  • 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:

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[edit]

File:Dinh 2009-11-25 Bisulfite Converted Jurket gDNA verification.jpg

C. November 25, 2009 - Capturing Protocol CpG30K[edit]

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[edit]

Added 1ul SLN Mix

Reagent Stock Conc Final Conc 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[edit]

Added 2ul ExoI/III mix to each rxn.

Dec 2 2009 PCR Amplification of Capture Products[edit]

PCR Amplification

Reagent Vol (1x) 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

File:Dinh Capture reaction after PCR amp.jpg