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''' | ||
Line 49: | Line 50: | ||
:- ''Incubate at 37C for 3hr'' | :- ''Incubate at 37C for 3hr'' | ||
<br> | <br> | ||
=== | === 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- | ||
Line 119: | Line 120: | ||
| H2O||||||||||10.60||127.20 | | H2O||||||||||10.60||127.20 | ||
|} | |} | ||
:- Aliquot 40ul, add | :- Aliquot 40ul, add 10ul of DNA template<br> | ||
::- 95C for 2min --> [95C for 20sec -> '''60C for 30sec''' -> 72C for 1min] X 25 cycles --> 72C for 2min<br> | ::- 95C for 2min --> [95C for 20sec -> '''60C for 30sec''' -> 72C for 1min] X 25 cycles --> 72C for 2min<br> | ||
:- I took 4ul of the first round PCR amplicons to verify in the gel | :- I took 4ul of the first round PCR amplicons to verify in the gel | ||
Line 140: | Line 141: | ||
[[File:ZhangLab_2 2014-02-15 16hr 40min_1stround_Exp2_3ul-of-33_after-bead.jpg| 450px]] | [[File:ZhangLab_2 2014-02-15 16hr 40min_1stround_Exp2_3ul-of-33_after-bead.jpg| 450px]] | ||
* From the gel image, the adapters were not completely remove by bead purification. | * From the gel image, the adapters were not completely remove by bead purification. | ||
==== 2nd round amplification ==== | |||
:{| {{table}} border = 1 | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''Conc''' | |||
| align="center" style="background:#f0f0f0;"|'''unit''' | |||
| align="center" style="background:#f0f0f0;"|'''Final conc.''' | |||
| align="center" style="background:#f0f0f0;"|'''unit''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''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<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 at cycle 10 and 12 since qPCR curves have dropped or saturated for all reactions even NTC. | |||
==== PAGE analysis ==== | |||
:- Loaded 4ul of PCR product in 6% TBE gel | |||
[[File:ZhangLab_2 2014-02-15 17hr 16min_2ndround_Exp2_10cycles.jpg| 450px]] | |||
[[File:ZhangLab_2 2014-02-15 17hr 16min_2ndround_Exp2_12cycles.jpg| 450px]] | |||
* As expected the majority of amplified products derived from primer dimers and adapter | |||
''' DISCUSSION WILL BE ADDED''' | |||
* Continued on: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2014-2-17]] |
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
- - 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
- - 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]]