User:Karmella Haynes/Notebook/PcTF Genomics/2014/06/04: Difference between revisions
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==06/04/14== | ==06/04/14== | ||
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* 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 | ||
* | * 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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|} | |} | ||
* Conclusion: discard samples | * Conclusion: <font color="red">discard samples</font> | ||
* Do a new RNA extraction from TRIzol 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'''<br> | |||
{| class="wikitable" border="0" cellspacing="5" | |||
|- | |||
| <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 || 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''' | '''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 = | * Total reactions = 12 <!-- Replace N with the total number of reactions! --> | ||
* Samples: | * Samples: | ||
# K562 | # K562 1 (E004) | ||
# K562 1 | # K562 1 (E005) | ||
# K562 2 | # K562 1 (E006) | ||
# K562 2 | # 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 ( | | <u>Reagent</u> || <u>Single rxn.</u> || Mix (x12) | ||
|- | |- | ||
| 10x RT buffer || 2.0 || | | 10x RT buffer || 2.0 || 24.0 | ||
|- | |- | ||
| 25 mM MgCl<sub>2</sub> || 4.0 || | | 25 mM MgCl<sub>2</sub> || 4.0 || 48.0 | ||
|- | |- | ||
| 0.1 M DDT || 2.0 || | | 0.1 M DDT || 2.0 || 24.0 | ||
|- | |- | ||
| RNaseOUT || 1.0 || | | RNaseOUT || 1.0 || 12.0 | ||
|- | |- | ||
| SuperScript III RT || 1.0 || | | SuperScript III RT || 1.0 || 12.0 | ||
|- | |- | ||
| || 10.0 μL || | | || 10.0 μL || 120.0 μL | ||
|} | |} | ||
Revision as of 20:25, 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> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
06/04/14
cDNA synthesis: K562
--> Incubate at 65°C/ 5 min. Immediately place on ice for 1 min.
--> Aliquot 10 μL of mix into 8-tube strip
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