User:Karmella Haynes/Notebook/PcTF Genomics/2014/06/04: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
Line 19: Line 19:




'''Measure concentrations'''
'''Measure RNA concentration'''<br>
{| class="wikitable" border="0" cellspacing="3" <!-- DNA concentration table -->
 
|-valign="top"
{| class="wikitable" border="0" cellspacing="5"
| <u>Sample</u> || <u>OD 260</u> || <u>260/280</u> || <u>ng/μL</u>
|-
| <u>Sample</u> || <u>OD 260</u> || <u>260/280</u> || <u>ng/μL</u> || <u>Vol. to use for cDNA synth.</u>
|-
| 1. ###    || ### || ### || ### || ### μL (### μg)
|-
| 2. ### || ### || ### || ### || ### μL (### μg)
|}
 
 
'''oligo(dT) Primer annealing'''
 
{| class="wikitable" border="0" cellspacing="5"
|-
| <u>Reagent</u>        || <u>Vol</u>
|-
| total RNA (up to 2μg) || up to 8 μL
|-
|-
| K562 1.1 || ### || ### || ###
| 50 μM oligo(dT) primer || 1.0
|-
|-
| K562 2.1 || ### || ### || ###
| 10 mM dNTP mix        || 1.0
|-
| DEPC-treated water    || = 8.0 - vol. total RNA
|-
| &nbsp;                || 10.0 μL
|}
|}


--> Incubate at 65°C/ 5 min. Immediately place on ice for 1 min.


'''cDNA synthesis'''
* SuperScript III


'''cDNA synthesis mix'''
* Total reactions = 4 <!-- Replace N with the total number of reactions! -->
* Samples:
# K562 1.1 (E004)
# K562 1.1 (E004)
# K562 1.1 (E005)
# K562 1.1 (E005)
Line 38: Line 60:
# K562 2.1 (C005)
# K562 2.1 (C005)


{| class="wikitable" border="0" cellspacing="3" <!-- Rxn. table -->
{| class="wikitable" border="0" cellspacing="5"
|-valign="top"
| <u>Reagent</u> || <u>Volume</u>
|-
|-
| reagent 1 || # μL
| <u>Reagent</u>        || <u>Single rxn.</u> || Mix (x4)
|-
|-
| reagent 2 || #
| 10x RT buffer          || 2.0        || 8.0
|-
|-
| reagent 3 || #
| 25 mM MgCl<sub>2</sub> || 4.0        || 16.0
|-
|-
| reagent 4 || #
| 0.1 M DDT              || 2.0        || 8.0
|-
|-
| dH<sub>2</sub>O || #
| RNaseOUT              || 1.0        || 4.0
|-
| SuperScript III RT    || 1.0        || 4.0
|-
|-
| &nbsp; || # μL
| &nbsp;                 || 10.0 μL    || 40.0 μL  
|}
|}
--> Aliquot 10 μL of mix into 8-tube strip<br>
--> Add annealing rxn. into each 10 μL aliquot<br>
--> PCR machine: 50°C/ 50 min., 80°C/ 5 min., ice<br>
--> Add 1.0 μL RNase H, incubate at 37°C/ 20 min.<br>
--> Store at -20°C




Revision as of 12:41, 4 June 2014

Pc-TF Genomics <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>


06/04/14

  • cDNA synthesis: K562 sets ## - ##

cDNA synthesis: K562

  • Use Carly's RNA from -80 °C
  • Assume that K562 1.1 is +PcTF and K562 2.1 is control (will find out when QC plate for mCh RT-PCR is run)
  • Will hold off on continuing U2OS for transiently transfected samples; will switch to KAH126 for further analyses


Measure RNA concentration

Sample OD 260 260/280 ng/μL Vol. to use for cDNA synth.
1. ### ### ### ### ### μL (### μg)
2. ### ### ### ### ### μL (### μg)


oligo(dT) Primer annealing

Reagent Vol
total RNA (up to 2μg) up to 8 μL
50 μM oligo(dT) primer 1.0
10 mM dNTP mix 1.0
DEPC-treated water = 8.0 - vol. total RNA
  10.0 μL

--> Incubate at 65°C/ 5 min. Immediately place on ice for 1 min.


cDNA synthesis mix

  • Total reactions = 4
  • Samples:
  1. K562 1.1 (E004)
  2. K562 1.1 (E005)
  3. K562 2.1 (C004)
  4. K562 2.1 (C005)
Reagent Single rxn. Mix (x4)
10x RT buffer 2.0 8.0
25 mM MgCl2 4.0 16.0
0.1 M DDT 2.0 8.0
RNaseOUT 1.0 4.0
SuperScript III RT 1.0 4.0
  10.0 μL 40.0 μL

--> Aliquot 10 μL of mix into 8-tube strip
--> Add annealing rxn. into each 10 μL aliquot
--> PCR machine: 50°C/ 50 min., 80°C/ 5 min., ice
--> Add 1.0 μL RNase H, incubate at 37°C/ 20 min.
--> Store at -20°C





Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Karmella_Haynes/Notebook/PcTF_Genomics/2014/06/04&oldid=795924"