Noi/NOTES/2014-12-20: Difference between revisions

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>Noi
(Created page with "= scRBS experiment of single mouse neuronal nucleus (Exp #2) = '''Back to calendar'''<br> * This experiment continued from the the first of exper...")
 
>Noi
mNo edit summary
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* I had some discussion in previous experiment. For this experiment, I will MONITOR PCR in the 1st PCR carefully to prevent over-amplification of adaptor dimers.  
* I had some discussion in previous experiment. For this experiment, I will MONITOR PCR in the 1st PCR carefully to prevent over-amplification of adaptor dimers.  
* In addition, I will include more control either positive control and negative control in every step to make sure that where the signal comes from. I will also include and exclude unmeth lambda DNA as well.
* In addition, I will include more control either positive control and negative control in every step to make sure that where the signal comes from. I will also include and exclude unmeth lambda DNA as well.
== Sample list in brief in each step ==
== Sample list in each step in brief ==
==== Cell lysis ====
==== Cell lysis ====
:- WT_1 to WT_16 (WT 1 nu, performed by Youjin). I re-run the number of samples to make it easy to handle and track.
:- WT_1 to WT_16 (WT 1 nu, performed by Youjin). I re-run the number of samples to make it easy to handle and track.
Line 13: Line 13:
:- NTC with unmeth lambda DNA (duplicate),
:- NTC with unmeth lambda DNA (duplicate),
* All samples in this step will have 60fg of unmeth lambda DNA spike in. This allows for bisulfite conversion efficiency measurement.
* All samples in this step will have 60fg of unmeth lambda DNA spike in. This allows for bisulfite conversion efficiency measurement.
:- I did serial dilution of CTT gDNA from 2ng/ul to 100pg/ul and 5pg/ul
::- Mix 10ul of 2ng/ul CTT gDNA with 190ul H2O
::- Mix 10ul of 100pg/ul CTT gDNA with 190ul H2O
==== DNA fragmentation with MspI ====
==== DNA fragmentation with MspI ====
:- Include
:- 10pg of CTT '''without''' unmeth lambda DNA (duplicates).
:- MspI NTC  '''without''' unmeth lambda DNA (duplicates).
==== Methylated adaptor ligation ====
:- Ligation control (duplicates).
* No NTC during Gap-filling/dA-tailing
== Experimental procedure ==
* I used mouse neuronal nuclei picked on 2014-12-17 and stored at -80C
=== 2) Cell lysis ===
'''''<span style="color:crimson"><u>Prep</u></span>'''''<br>
- Thaw nuclei from -80C & spin down at 2,000rpm for 5min. I put PCR tube on PCR rack. (96-well plate rotor, 5min)<br>
- Mix QIAGEN Protease with lambda DNA. I prepared more than enough of protease and unmethy lambda DNA mix.
:{| {{table}} border = 1
| align="center" style="width:180px;background:#f0f0f0;"|'''Components'''
| align="center" style="width:80px;background:#f0f0f0;"|'''1X rxn'''
| align="center" style="width:80px;background:#f0f0f0;"|'''100X rxn'''
|-
| Cell in 1.43X lysis buffer||4.00||0.00
|-
| 1.2pg Lambda DNA||0.05||5.00
|-
| Protease||1.00||100.00
|-
| Total||5.05||
|}
* Final amount of unmet lambda DNA in each tube is 60fg.

Revision as of 09:57, 14 January 2015

scRBS experiment of single mouse neuronal nucleus (Exp #2)

Back to calendar

  • This experiment continued from the the first of experiment (Exp#1) for UCLA (Dr. Fan's group)
  • I had some discussion in previous experiment. For this experiment, I will MONITOR PCR in the 1st PCR carefully to prevent over-amplification of adaptor dimers.
  • In addition, I will include more control either positive control and negative control in every step to make sure that where the signal comes from. I will also include and exclude unmeth lambda DNA as well.

Sample list in each step in brief

Cell lysis

- WT_1 to WT_16 (WT 1 nu, performed by Youjin). I re-run the number of samples to make it easy to handle and track.
For my part I will include all controls and also in the following steps
- T_1 to T_16 (TKO 1 nu, performed by Noi).
- T10, T100 and T0 (TKO 10nu, 100nu and 0nu, 1 each, performed by Noi)
- 10pg of CTT with unmeth lambda DNA (duplicates). CTT is gDNA from frozen colon tumor tissue purchased from Biochain.
- NTC with unmeth lambda DNA (duplicate),
  • All samples in this step will have 60fg of unmeth lambda DNA spike in. This allows for bisulfite conversion efficiency measurement.
- I did serial dilution of CTT gDNA from 2ng/ul to 100pg/ul and 5pg/ul
- Mix 10ul of 2ng/ul CTT gDNA with 190ul H2O
- Mix 10ul of 100pg/ul CTT gDNA with 190ul H2O

DNA fragmentation with MspI

- 10pg of CTT without unmeth lambda DNA (duplicates).
- MspI NTC without unmeth lambda DNA (duplicates).

Methylated adaptor ligation

- Ligation control (duplicates).
  • No NTC during Gap-filling/dA-tailing

Experimental procedure

  • I used mouse neuronal nuclei picked on 2014-12-17 and stored at -80C

2) Cell lysis

Prep
- Thaw nuclei from -80C & spin down at 2,000rpm for 5min. I put PCR tube on PCR rack. (96-well plate rotor, 5min)
- Mix QIAGEN Protease with lambda DNA. I prepared more than enough of protease and unmethy lambda DNA mix.

Components 1X rxn 100X rxn
Cell in 1.43X lysis buffer 4.00 0.00
1.2pg Lambda DNA 0.05 5.00
Protease 1.00 100.00
Total 5.05
  • Final amount of unmet lambda DNA in each tube is 60fg.