Noi/NOTES/2014-2-14: Difference between revisions

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= scRRBS trial experiment #2 =
= scRRBS trial experiment #2 =
* Link to calendar: [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes#2014]]
* Link to calendar: [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes#2014]]
* Continued from: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2014-2-13]]
* 2014-02-13: Have flow sorted nuclei from Blue/Rui (3 devisable 96-well plates with the following layout). To prepare plate for nuclei sorting, 4ul of 1.2X lysis buffer without protease and unmethylated lambda DNA was added to each well.
* 2014-02-13: Have flow sorted nuclei from Blue/Rui (3 devisable 96-well plates with the following layout). To prepare plate for nuclei sorting, 4ul of 1.2X lysis buffer without protease and unmethylated lambda DNA was added to each well.
::{| {{table}}
::{| {{table}} class = wikitable
| align="center" style="background:#f0f0f0;" width="50"|''''''
| align="center" style="background:#f0f0f0;" width="50"|''''''
| align="center" style="background:#f0f0f0;" width="50"|'''1'''
| align="center" style="background:#f0f0f0;" width="50"|'''1'''
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:- ''Incubate at 37C for 3hr''
:- ''Incubate at 37C for 3hr''
<br>
<br>
=== Gap-filling/dA tailing ===
=== End-repair/dA-tailing ===
:- Add 1ul of dA/dC/dG mix
:- Add 1ul of dA/dC/dG mix
:- Add 1ul of Klenow fragment exo-
:- Add 1ul of Klenow fragment exo-
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:- Aliquot 20ul, add 30ul of DNA template<br>
:- Aliquot 20ul, add 30ul of DNA template<br>
::- 98C for 2min --> [98C for 10sec -> '''65C for 30sec''' -> 72C for 1min] X 22 cycles --> 72C for 2min<br>
::- 98C for 2min --> [98C for 10sec -> '''65C for 30sec''' -> 72C for 1min] X 22 cycles --> 72C for 2min<br>
:- I monitored qPCR and have taken 4ul of PCR product 15 cycle 10 and 12 since qPCR curves have dropped or saturated for all reactions even NTC.
:- I monitored qPCR and have taken 4ul of PCR product at cycle 10 and 12 since qPCR curves have dropped or saturated for all reactions even NTC.
 
==== PAGE analysis ====
==== PAGE analysis ====
:- Loaded 4ul of PCR product in 6% TBE gel
:- Loaded 4ul of PCR product in 6% TBE gel

Latest revision as of 00:12, 21 February 2014

scRRBS trial experiment #2[edit]

  • Link to calendar: [[1]]
  • Continued from: [[2]]
  • 2014-02-13: Have flow sorted nuclei from Blue/Rui (3 devisable 96-well plates with the following layout). To prepare plate for nuclei sorting, 4ul of 1.2X lysis buffer without protease and unmethylated lambda DNA was added to each well.
' 1 2 3
A 1000 1 1
B 100 1 1
C 10 1 1
D 1 1 1
E 1 1 1
G 1 0 1
H 1 0 1
  • Started experiment by including the the first column with bulk nuclei (A1-C1: 1000, 100, 10) as control, D1-H1 with single nuclei, and G2 & H2 as negative control.

Cell lysis[edit]

- Thaw nuclei from -20C & spin down
- Mix protease and unmethylated lambda DNA 1:1 ratio (15ul + 15ul). This will be used 1ul for nuclei lysis.
- Add 1ul of protease/unmethylated lambda DNA mix
- Incubate at 50C for 3hr
- Heat inactivate at 75C for 30min


Msp I digestion[edit]

Components Volume (ul) 12x rxn mix
Lysed nuclei 5.00 0.00
10X Tango buffer 2.00 24.00
MspI 0.90 10.80
H2O 10.10 121.20
Total 18.00 117.00
- Aliquot 13ul to each tube
- Incubate at 37C for 3hr


End-repair/dA-tailing[edit]

- Add 1ul of dA/dC/dG mix
- Add 1ul of Klenow fragment exo-
- Incubate at 30C for 15min
- Heat inactivate at 75C for 10 min**. (This step was not mentioned in paper.This time, I SKIPPED this step.)


Methylated adapter ligation[edit]

  • Used the same amount of methylated adapter as in previous experiment of 2014-02-13
Components Volume (ul) 12x rxn mix
dA-tailed reaction 20.00 0.00
10X Tango buffer 0.50 6.00
HC T4 DNA ligase (30units/ul) 1.00 12.00
10mM ATP 1.25 15.00
H2O 1.25 15.00
Total 24.00 48.00
  • PEG 4000 was not added in this experiment
- Add 1ul of diluted methylated adapter
- Add 4ul of ligation reaction mix
- Mix by pipetting and spin down
- Incubate at 22C for 1hr


Bisulfite conversion[edit]

  • I performed bisulfite conversion using the same procedure as described on 2014-02-13 but eluted with larger volume, 32ul, as described in paper.
- Mix 25ul of DNA sample with 162.5ul of CT Conversion Reagent
- 98°C for 10 minutes (DNA denaturation)
- 64°C for 2.5 hours (Bisulfite conversion)
- 4°C storage for up to 20 hours or continue to desulfonation
- Bind DNA to column by mixing with 750ul Binding Buffer and 5ul of 2ng/ul tRNA as carrier. Spin down 14,000rpm for 30sec
- Wash with 100ul Wash buffer. Spin down 14,000rpm for 30sec
- Incubate with 200ul of Desulphonation Buffer for 15min. Spin down 14,000rpm for 30sec
- Wash column with 200ul Wash Buffer. Spin down 14,000rpm for 30sec. Discard spnt.
- Wash the column with 200ul Wash Buffer. Spin down 14,000rpm for minsec
- Elute converted DNA with warm (~60C) 32ul Elution Buffer. This should have ~30ul DNA left for PCR

PCR amplification[edit]

1st round amplification[edit]

Components Conc unit Final conc. unit Volume (ul) 12 rxn mix
Bis-cvt DNA 30.00 0.00
10X Reaction buffer 10 X 1 X 5.00 60.00
dNTP mix 10 mM 0.2 mM 1.00 12.00
PCR_F 10 uM 0.2 uM 1.00 12.00
PCR_N2.Ind2 10 uM 0.2 uM 1.00 12.00
PfuTurbo Cx 2.5 Unit/ul 1 unit 0.40 4.80
50X SYBG 50 X 1 X 1.00 12.00
H2O 10.60 127.20
- Aliquot 40ul, add 10ul of DNA template
- 95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 25 cycles --> 72C for 2min
- I took 4ul of the first round PCR amplicons to verify in the gel
File:ZhangLab 2 2014-02-15 16hr 00min 1stround Exp2 4ul.jpg 
- Perform two rounds AMPure bead purification

AMPure bead purification[edit]

- Add 46ul AMPure bead. Mix by pipetting 10x
- Sit for 8min
- Transfer to sit on magnet for 3min
- Wash twice with 150ul freshly prepared 80% EtOH
- Dry the bead for 3-5min
- Resuspend with 40 H2O
- Add 40ul AMPure bead. Mix by pipetting 10x (Note that, AMPure beads in the first round were saved and combined with the second round beads). In the future, I may only add binding buffer, 20%PEG/NaCl, in the second round of purification.
- Sit for 8min
- Transfer to sit on magnet for 3min
- Wash twice with 150ul freshly prepared 80% EtOH
- Dry the bead for 3-5min
- Resuspend with 33ul H2O
  • From PAGE analysis of the first round PCR amplicons above, the majority of amplified product were primer dimers (<50bp) and adapters (~125bp). I wanted too see how bead purification could remove those band, I took 3ul to verify in 6% TBE gel.
File:ZhangLab 2 2014-02-15 16hr 40min 1stround Exp2 3ul-of-33 after-bead.jpg
  • From the gel image, the adapters were not completely remove by bead purification.

2nd round amplification[edit]

Components Conc unit Final conc. unit Volume (ul) 12 rxn mix
Purified 1st round DNA 30.00 0.00
5X Phusion HF buffer 5 X 1 X 10.00 120.00
dNTP mix 10 mM 0.2 mM 1.00 12.00
PCR_F 10 uM 0.2 uM 1.00 12.00
PCR_N2.Ind2 10 uM 0.2 uM 1.00 12.00
50X SYBR 50 X 1 X 1.00 12.00
Phusion HF 2 unit/ul 0.50 6.00
H2O 5.50 66.00
- Aliquot 20ul, add 30ul of DNA template
- 98C for 2min --> [98C for 10sec -> 65C for 30sec -> 72C for 1min] X 22 cycles --> 72C for 2min
- I monitored qPCR and have taken 4ul of PCR product at cycle 10 and 12 since qPCR curves have dropped or saturated for all reactions even NTC.

PAGE analysis[edit]

- Loaded 4ul of PCR product in 6% TBE gel
File:ZhangLab 2 2014-02-15 17hr 16min 2ndround Exp2 10cycles.jpg
File:ZhangLab 2 2014-02-15 17hr 16min 2ndround Exp2 12cycles.jpg
  • As expected the majority of amplified products derived from primer dimers and adapter

DISCUSSION WILL BE ADDED

  • Continued on: [[3]]