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Yeast Colony PCR Troubleshooting

Yeast Colony PCR Troubleshooting - PCR - Polymerase Chain Reaction Forum

Yeast Colony PCR Troubleshooting - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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  #1  
Old 12-28-2007, 12:04 AM
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Unhappy Yeast Colony PCR Troubleshooting



Hey guys,


I have a problem with my yeast colony PCR.

I am including my yeast colony PCR protocol here:


1. Incubate yeast culture with medium at 30C, overnight.
2. Then centrifuge 0.5ml of cells at 8000rpm for 5mins, after discard supernatant.
3. Resuspend the cells with 0.5ml 1xPBS, centrifuge at 8000rpm for 5mins,discard supernatant.
4. Resuspend the cells with 0.1ml TE, then boil in water for 3 mins
5. cold in the liquid nitrogen for 0.5min
6. boiled in water for 2mins.
7.then take 1ul supernatant as template for PCR

The problem is that i cannot get the right PCR product, I actually get nothing.

Is this yeast colony protocol ok?

cheers
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Old 05-23-2008, 04:50 PM
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Exclamation Re: Yeast Colony PCR Troubleshooting

I have an alternative approach, and it works very well.
I just pick my colonies into 25ul of 20mM NaOH and heat them at 95C for 10min.


Last edited by admin; 02-15-2011 at 11:05 AM.
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  #3  
Old 05-24-2008, 09:27 PM
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Default Re: Yeast Colony PCR Troubleshooting

TE--shouldn't add EDTA to PCR-it's a taq inhibitor.
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Old 06-12-2008, 07:17 PM
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Default Re: Yeast Colony PCR Troubleshooting

Try this protocol:-

1- spin cells down and pour off supernatant.

2- wash with PBS (1x) and spin down. pour off supernatant.

3- resuspend in sdH2O. depending on culture density and volume. for a 1 mM diameter culture I resuspend in 50ul.

4- heat at 95C for 5 mins to lyse cells and deactivate DNases.

5- spin down at 12-14000 x to remove debris.

6- transfer supernatant to a fresh tube and that's your DNA for colony PCR.

goodluck
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