About PCR from a single yeast colony

Yang Sheng shengyang at USA.NET
Thu Oct 15 07:57:37 EST 1998


At 98-9-9 14:38:00, you wrote:
>In article <1C65015A0F31 at serverdos.cigb.edu.cu>, verena.muzio at cigb.edu.cu wrote:
>
>> Dear colleagues:
>> Could somebody send me a simple protocol for PCR from a single yeast
>> colony. 
>> Please, write to my e-mail address 
>> Thanking in advance,
>> Alexis
>
>Hi Alexis
>
>I do yeast colony PCR very often. I use 0.1% Triton-X in my PCR mix with no
>enzyme (taq) and set a 10min 92°C in the start of the PCR program to break
>the yeat cell membrane. When the 10min is about to finish I add the enzyme.
>This way U get more of the enzyme.
>
>Good luck,
>
>Mohammad
>      _ _
>_ooO_(o o)_Ooo_
>
Hi,
I am doing direct PCR screening of Pichia pastoris clones according to BioTechniques 20: 980-2 
(June 1996). However, having been treated by 5U/ul of lyticase or zymolyase, the cell pellet 
seemed not lysed. I wonder whether the step of freezing the samples at -80degree does the key 
work.

The brief protocol is as follows:
1. Pick a pinpoint-size part of a colony and suspend the cells in 10ul of water;
2. Add 5ul of a 5U/ul solution of Lyticase, mix and incubate at 30degree for 10';
3. Freeze the samples at -80degree for 10' or immerse them in liquid nitrogen for 1'; 
 	/* I used the latter.*/
4. Setup a 50ul PCR (hot start):
	10XReaction Buffer:					5ul
	MgCl2(25mM):						5ul
	dNTP mixtures (25mM each):				1ul
	Primer1 (e.g. AOX1 5' primer) (10pmol/ul):	1ul
	Primer1 (e.g. AOX1 3' primer) (10pmol/ul):	1ul
	H2O: 							     27ul
	Add 5ul of sample, mix and overlay with 20ul of mineral oil;
5. Put teh samples into the cycler and heat them at 95degree for 5'
   Add 5ul of 0.16U/ul solution (=0.8U) of Taq DNA polymerase
6. Cycle 30 times at the following conditions
	denaturation step: 	95, 1'
	annealing step:		54, 1'
	extension step:		72, 1'
	(for inserts up to 2000bp; with a final step of 10' extension at 72degree)
	 /* My inserts is 1900bp */
7. Run a 10ul aliquot of the reactions on agarose gel of appropriate percentage.
	 /* I found no band in the gel :-( */

Any idea?




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