Noi/NOTES/2014-2-14: Difference between revisions
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>Noi mNo edit summary |
>Noi mNo edit summary |
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| 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 | ||
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[[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 15 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''' |
Revision as of 21:31, 19 February 2014
scRRBS trial experiment #2
- Link to calendar: [[1]]
- 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
- - 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
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
Gap-filling/dA tailing
- - 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
- 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
- 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
1st round amplification
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
- - 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
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 15 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
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