AlanFung:LabNotes/CTCF/2011-5-10: Difference between revisions

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Line 26: Line 26:
| align="center" style="background:#f0f0f0;"|'''Volume'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
|-
|100% Ethanol||align="right"|2.5X Sample Volume||align="right"|6000uL
|100% Ethanol||align="right"|2.5X Sample Volume||align="right"|950uL
|-
|-
|3M NaOAC||align="right"|0.1X Sample Volume||align="right"|240uL
|3M NaOAC||align="right"|0.1X Sample Volume||align="right"|38uL
|-
|-
|Glycoblue||align="right"|1/300X Sample Volume||align="right"|8uL
|Glycoblue||align="right"|1/300X Sample Volume||align="right"|2uL
|-
|-
|Sample||align="right"|Sample Volume||align="right"|2400uL
|Sample||align="right"|Sample Volume||align="right"|380uL
|-
|-
|Total Volume
|Total Volume
|colspan="2" align="right"|8648uL
|colspan="2" align="right"|1370uL
|-
|-
|}
|}
Line 49: Line 49:


===Qubit Quantifiction===
===Qubit Quantifiction===
*CHR8-1.87ng/ul
*CHR8-1.87ng/ul 15.37nM
*CHR21-0.545ng/ul
*CHR21-0.545ng/ul 4.48nM
*CHR22-1.12ng/ul
*CHR22-1.12ng/ul 9.21nM


===Determine the efficiency of bisulfite conversion===
===Determine the efficiency of bisulfite conversion===
Line 57: Line 57:


===Make Serial Dilution on Control DNA===
===Make Serial Dilution on Control DNA===
*Create 6 STDs
*number of copies = ( amount * 6.022x1023) / (length * 1x10^9 * 650)
*Calculation for STD based on 200bp and 8ul of the dsDNA
*Create 4 STDs
*1:1000 dilution from 20pM to 0.02pM
*1:1000 dilution from 0.02pm to 0.00002pM (this is STD1)
{| {{table}}
{| {{table}}
| Sample Name||dsDNA Concentration (pM)
| align="center" style="background:#f0f0f0;"|'''Sample Name'''
| align="center" style="background:#f0f0f0;"|'''Concentration (pM)'''
| align="center" style="background:#f0f0f0;"|'''Amount (ng)'''
| align="center" style="background:#f0f0f0;"|'''DNA Molecule'''
|-
|-
| Std 1||20
| STD1||0.00002||1.94E-05||90100
|-
|-
| Std 2||2
| STD2||0.000002||1.94E-06||9010
|-
|-
| Std 3||0.2
| STD3||0.0000002||1.94E-07||901
|-
|-
| Std 4||0.02
| STD4||0.00000002||1.94E-08||90.1
|}
*With starting amount of 600pg, assume the % lost of sample is 90, 10 and 1. The # of molecules of resulting DNA will be as follows.
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''%'''
| align="center" style="background:#f0f0f0;"|'''ng'''
| align="center" style="background:#f0f0f0;"|'''DNA Molecule'''
|-
| 100||0.6||86.9
|-
|-
| Std 5||0.002
| 90||0.54||78.2
|-
|-
| Std 6||0.0002
| 10||0.06||8.69
|-
|-
|  
| 1||0.006||0.869
|}
|}



Latest revision as of 17:07, 2 June 2011

Bisulfite Conversion DNA Damage Estimation[edit]

  • Perform PCR with CHR8, CHR21 and CHR22
  • These will be our standards

File:ZhangLab 2 2011-05-10 10hr 40min.jpg

  • Need to purify CHR8, CHR21, and CHR22.
    • Qubit Results(ng/ul)
    • Assume amplicon length ~200bp
  • CHR8-3.25ng/ul 26.72nM
  • CHR21-4.39ng/ul 36.09nM
  • CHR22-4.77ng/ul 39.21nM
  • It is better to perform a size selection, since there are some ghosting on the gel

Size Selection[edit]

File:ZhangLab 2 2011-05-26 17hr 09min.jpg

  • Prepare 3 gel shearing assemblies by placing a 0.5 mL tube within another 1.5 mL Non-Stick tube. Use G20 needle to punch a small hole at the bottom of the 0.5 mL tube. Use two assemblies for each PAGE gel.
  • Cut out the correct band (~200 nt in this case), transfer gel slice to shearing assemblies
  • Centrifuge at top speed > 12000 rpm for 2 min to sheer the gel, remove the 0.5 mL tube.
  • Add 400 uL clean 1x TE buffer in the 1.5 mL tube. Tape the tubes on a vortexer, place the vortexer in 37C incubator, shake the tubes at low speed for 1 hour. Alternatively, can keep the tubes in 4C fridge overnight.
  • Spin down all tubes at 13,000rpm for 2min
  • In the PCR hood, transfer the liquid layer of the gel mix to Nanosep columns, spin for 3 min at top speed (15,000 rpm), and then transfer supernatant to 1.5mL tube.
  • Perform ethanol precipitation
  • Add in required volume of 100% ethanol, 3M NaOAC and Glycoblue into tray
Content Volume Needed Volume
100% Ethanol 2.5X Sample Volume 950uL
3M NaOAC 0.1X Sample Volume 38uL
Glycoblue 1/300X Sample Volume 2uL
Sample Sample Volume 380uL
Total Volume 1370uL
  • Mix all content by repeat pipetting using multichannel pipette
  • Store in -80C for more than 20 mins or forever
  • Turn on Centrifuge fix temperature at 4C, place a tube of 75% ethanol into 4C fridge
  • Centrifuge frozen 1.5mL tubes at 10000rpm for 30m at 4C
  • Remove all liquid CAUTION: DO NOT DISCARD BLUE PELLET AT THE BOTTOM OF THE TUBE
  • Add in 750uL cold 75% Ethanol, mix by repeat pipetting and transfer to a 1.5mL tube
  • Centrifuge at 10,000rpm for 5min at 4C
  • Remove all liquid, air dry sample for 5min or until samples dried out
  • Resuspend the DNA with 20uL RNAse free H2O

Qubit Quantifiction[edit]

  • CHR8-1.87ng/ul 15.37nM
  • CHR21-0.545ng/ul 4.48nM
  • CHR22-1.12ng/ul 9.21nM

Determine the efficiency of bisulfite conversion[edit]

  • After performing the bisulfite conversion procedures, we should perform real-time PCR of the samples and controls to determine the amount of converted human genomic DNA. Base on the we can determine the conversion efficiency.

Make Serial Dilution on Control DNA[edit]

  • number of copies = ( amount * 6.022x1023) / (length * 1x10^9 * 650)
  • Calculation for STD based on 200bp and 8ul of the dsDNA
  • Create 4 STDs
  • 1:1000 dilution from 20pM to 0.02pM
  • 1:1000 dilution from 0.02pm to 0.00002pM (this is STD1)
Sample Name Concentration (pM) Amount (ng) DNA Molecule
STD1 0.00002 1.94E-05 90100
STD2 0.000002 1.94E-06 9010
STD3 0.0000002 1.94E-07 901
STD4 0.00000002 1.94E-08 90.1
  • With starting amount of 600pg, assume the % lost of sample is 90, 10 and 1. The # of molecules of resulting DNA will be as follows.
% ng DNA Molecule
100 0.6 86.9
90 0.54 78.2
10 0.06 8.69
1 0.006 0.869

Prepare qPCR Reagents[edit]

Ensure that the following reagents are completely thawed and thoroughly mixed by vortexing[edit]

  • 2X KAPA SYBR FAST qPCR Master Mix
  • 6 DNA Standards
  • 1:1000 dilution of bisulfite converted library DNA
  • Primer Mix (F+R) 1:1 ratio 1.65uM

Prepare qPCR[edit]

CHR8 CHR21 CHR22
2X iQ Super Mix 20 20 20
Primer F+R (1.65uM) 4.85 4.85 4.85
gDNA 8 8 8
ddH2O 7.15 7.15 7.15
Total 40 40 40

Perform real-time PCR reaction in thermocycler[edit]

      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

Generate Standard Curve[edit]

For Bio-Rad Real time Thermal Cycler[edit]

  • Edit plate setup
  • Select and add wells with control DNA as Standard
  • Specify Quant Standards (bottom right button)
  • After PCR cycle
  • Select Quantitation Tab on left column to see standard curve
  • Select Calculations Tab at the bottom to see the sample concentrations

For Eppedorf Real time Thermal Cycler[edit]

  • Edit Plate Layout
  • Select wells for standards
  • right click on well and select well as standard
  • Input name and standard amount
  • After PCR cycle
  • Standard curve will show up on the bottom half of the analysis screen
  • Sample concentrations will show up on left column

Generate Recovery Curve[edit]