#Prepare PCR:
===Run PCR Program Colony PCR:===
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==PCR Program==
[[ Category:PCR]]
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*Prepare gel 30 minutes before PCR is finished.
*Add this mixture to each PCR tube.
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*DreamTaq Green PCR Master mix - contains dNTPs, polymerase, salts, and buffer with loading dye
==PCR Program==
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*SyberGreen PCR Master Mix Applied Biosystems
===RT-PCR Reaction===
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Run "specfic" PCR Program for gene of interest (approx 2 hours).
*[[PCR Amplification of DNA]]
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*Run PCR Program (approx 3.5 to 4 hours). Normally use touchdown PCR ('''TD-KOD''') as follows:
*Purify PCR product if necessary using Qiagen kit (Add 5x PB)
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* Platinum PCR SuperMix High Fidelity (Life Technologies cat# 12532-016)
* The PCR program should be
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# PCR product size should be set to 70-150bp.
[[ Category: PCR ]]
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Make primers at 1uM (Fwd + Rev and Fwd + Neo Rev}. PCR primers separately.
==PCR PROTOCOL==
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#Prepare PCR:
===Run PCR Program Colony PCR:===
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Make primers at 1uM (Fwd + Rev and Fwd + Neo Rev). PCR primers separately.
==PCR PROTOCOL==
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Make primers at 0.4uM (Fwd1 + Rev and Fwd2 + Rev). PCR primers separately.
==PCR PROTOCOL==
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[[ Category:PCR]]
==PCR Program==
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==PCR Program==
[[ Category:PCR]]
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==PCR PROTOCOL==
[[ Category:PCR]]
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*DreamTaq Green PCR Master mix - contains dNTPs, polymerase, salts, and buffer with loading dye
==PCR Program==
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[[Category: PCR ]]
*Get sequences from Xiao-ling, stocks in XL PCR Box
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*Prepare gel 30 minutes before PCR is finished.
*Add this mixture to each PCR tube.
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[[ Category:PCR]]
==PCR Program==
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#PCR desired insert using Pfu polymerase. Add 1 uL of Taq and mix. Incubate 10
#If PCR product is a single band, no need to purify further. If several bands, gel
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[[Category:PCR]]
...[[Rapid Isolation of Genomic DNA from Yeast]]. Use 1µl of the DNA in your PCR reaction.
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==PCR Reaction==
The PCR program is called '''ob-ob''' and is:
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...sc1 and the deleted allele using the following three primers in a 35 cycle PCR reaction using Perkin Elmer AmpliTaq Gold: F4536, 5′-AGGAGGCCTCTTCTGCTACC-3
[[ Category:PCR]]
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==PCR Reaction==
The PCR program is called "srebp" on the thermal cycler and is as follows:
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|[[TSC-floxed Genotyping| TSC-floxed]]|| [[Cre PCR]] || KO- 368bp, WT- 295bp || aTSC, mTSC, Placenta TSC || Two bands if heter
|[[Cre Genotyping|Cre]] || [[Cre PCR ]] || Between 340-400kDa || aTSC, mTSC, Adipo-Raptor, Adipo-GR-KO, Placenta
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*set up a 250 uL PCR reaction
**15 uL MgCl (this can be adjusted to optimize PCR conditions)
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* Platinum PCR SuperMix High Fidelity (Life Technologies cat# 12532-016)
* The PCR program should be
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*SyberGreen PCR Master Mix Applied Biosystems
===RT-PCR Reaction===
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