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

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Pc-TF Genomics</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Pc-TF Genomics</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
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==06/04/14==
==06/04/14==
<!-- Precede finished items with a checkmark &#x2713; -->
<!-- Precede finished items with a checkmark &#x2713; -->
* cDNA synthesis: K562 sets ## - ##
* cDNA synthesis: K562 sets 4 - 9
 


----
----
'''cDNA synthesis: K562'''<br>
'''cDNA synthesis: K562'''<br>
* Use Carly's RNA from -80 °C
* 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)
* K562 1 and 3 are +PcTF and K562 2 and 4 are controls (see [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/PcTF_Genomics/2012/12/18 12/18/12] ...will confirm 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
* Will hold off on continuing U2OS for transiently transfected samples; will switch to KAH126 for further analyses


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'''RNA extraction'''
'''New RNA extraction'''
* Follow the TRIzol/ RNeasy protocol
* Follow the TRIzol/ RNeasy protocol
* Use Carly's TRIzol samples K562 1, 2, 3, and 4
* Use Carly's TRIzol samples K562 1, 2, 3, and 4
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| <u>Sample</u> || <u>OD 260</u> || <u>260/280</u> || <u>ng/μL</u> || <u>Vol. to use for cDNA synth.</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. K562 1     || 0.975 || 2.017 || 780.25 || ### μL (2.5 μg)
| 1. K562 1 || 0.975 || 2.017 || 780.25 || 3.2 μL (2.5 μg)
|-
|-
| 2. K562 2  || 0.890 || 2.021 || 711.81 || ### μL (2.5 μg)
| 2. K562 2  || 0.890 || 2.021 || 711.81 || 3.5 μL (2.5 μg)
|-
|-
| 2. K562 3  || 0.856 || 2.028 || 684.53 || ### μL (2.5 μg)
| 2. K562 3  || 0.856 || 2.028 || 684.53 || 3.7 μL (2.5 μg)
|-
|-
| 2. K562 4  || 0.696 || 2.020 || 556.52 || ### μL (2.5 μg)
| 2. K562 4  || 0.696 || 2.020 || 556.52 || 4.5 μL (2.5 μg)
|}
|}
 
* Conclusion: Success! Yields are very good. Continue with cDNA synthesis, 3 reactions per RNA.




'''oligo(dT) Primer annealing'''
'''oligo(dT) Primer annealing'''
{| class="wikitable" border="0" cellspacing="5"
{| class="wikitable" border="0" cellspacing="5"
|-
|-
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'''cDNA synthesis mix'''
'''cDNA synthesis mix'''
* Total reactions = 4 <!-- Replace N with the total number of reactions! -->
* Total reactions = 12 <!-- Replace N with the total number of reactions! -->


* Samples:
* Samples:
# K562 1.1 (E004)
# K562 1 (E004)
# K562 1.1 (E005)
# K562 1 (E005)
# K562 2.1 (C004)
# K562 1 (E006)
# K562 2.1 (C005)
# K562 2 (C004)
# K562 2 (C005)
# K562 2 (C006)
# K562 3 (E007)
# K562 3 (E008)
# K562 3 (E009)
# K562 3 (C007)
# K562 3 (C008)
# K562 3 (C009)


{| class="wikitable" border="0" cellspacing="5"
{| class="wikitable" border="0" cellspacing="5"
|-
|-
| <u>Reagent</u>        || <u>Single rxn.</u> || Mix (x4)
| <u>Reagent</u>        || <u>Single rxn.</u> || Mix (x12)
|-
|-
| 10x RT buffer          || 2.0        || 8.0
| 10x RT buffer          || 2.0        || 24.0
|-
|-
| 25 mM MgCl<sub>2</sub> || 4.0        || 16.0
| 25 mM MgCl<sub>2</sub> || 4.0        || 48.0
|-
|-
| 0.1 M DDT              || 2.0        || 8.0
| 0.1 M DDT              || 2.0        || 24.0
|-
|-
| RNaseOUT              || 1.0        || 4.0
| RNaseOUT              || 1.0        || 12.0
|-  
|-  
| SuperScript III RT    || 1.0        || 4.0
| SuperScript III RT    || 1.0        || 12.0
|-
|-
| &nbsp;                || 10.0 μL    || 40.0 μL  
| &nbsp;                || 10.0 μL    || 120.0 μL  
|}
|}


Latest revision as of 00:01, 27 September 2017

Pc-TF Genomics Main project page
Previous entry      Next entry


06/04/14

  • cDNA synthesis: K562 sets 4 - 9


cDNA synthesis: K562

  • Use Carly's RNA from -80 °C
  • K562 1 and 3 are +PcTF and K562 2 and 4 are controls (see 12/18/12 ...will confirm 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. K562 1.1 -0.006 1.968 -5.193 n/a
2. K562 2.1 -0.003 1.6 -2.724 n/a
  • Conclusion: discard samples
  • Do a new RNA extraction from TRIzol samples


New RNA extraction

  • Follow the TRIzol/ RNeasy protocol
  • Use Carly's TRIzol samples K562 1, 2, 3, and 4


Measure RNA concentration

Sample OD 260 260/280 ng/μL Vol. to use for cDNA synth.
1. K562 1 0.975 2.017 780.25 3.2 μL (2.5 μg)
2. K562 2 0.890 2.021 711.81 3.5 μL (2.5 μg)
2. K562 3 0.856 2.028 684.53 3.7 μL (2.5 μg)
2. K562 4 0.696 2.020 556.52 4.5 μL (2.5 μg)
  • Conclusion: Success! Yields are very good. Continue with cDNA synthesis, 3 reactions per RNA.


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 = 12
  • Samples:
  1. K562 1 (E004)
  2. K562 1 (E005)
  3. K562 1 (E006)
  4. K562 2 (C004)
  5. K562 2 (C005)
  6. K562 2 (C006)
  7. K562 3 (E007)
  8. K562 3 (E008)
  9. K562 3 (E009)
  10. K562 3 (C007)
  11. K562 3 (C008)
  12. K562 3 (C009)
Reagent Single rxn. Mix (x12)
10x RT buffer 2.0 24.0
25 mM MgCl2 4.0 48.0
0.1 M DDT 2.0 24.0
RNaseOUT 1.0 12.0
SuperScript III RT 1.0 12.0
  10.0 μL 120.0 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





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