发信人: tops (digging), 信区: Biology
标 题: Re: 哪位给贴个用PCR检查阳性克隆的protocol吧
发信站: The unknown SPACE (Fri May 23 19:37:52 2003), 站内信件


1. Prepare PCR tubes with 5ul water in each;
2. prepare 15ml falcon tubes with 1ml of LB + antibiotics;
3. pick a colony using a sterile tip; stir it in a the water
-containing PCR tube for several times and then throw the
tip in the falcon tube to inoculate the LB medium.
4. use a primer on the vector and a primer on your insert
to prepare 15ul master mix for each 20ul reaction.
By using this primer set, you will be able to confirm
both availibility of insert and its orientation.
5. add 15ul master mix to each above PCR tube.
Use this condition for the PCR:
[95C, 5'] [95C,30"; 55C, 30"; 72C,1'/kb]25cycles [72C,2']
95C,5' will break the bacterial cells.

After you run the agarose gel, you wil be able to throw out
some negative LB cultures in falcon tubes before you do miniprep.
Or, you may use positive culture to inoculate a midiprep culture.

【 在 golem (小岩鼠) 的大作中提到: 】
: 比较省事的就好.


--
实验总是做不完的,
老婆可只有一个...

※ 来源:．The unknown SPACE bbs.mit.edu．[FROM: 198.151.]