H****s
发帖数: 301 | 1 各位同学,谁有Yeast Colony PCR protocol分享一下? 现在用的这个太费时间,费精力,
并且要好几次才能做出来.谢谢啦. | X******n
发帖数: 914 | 2 For plasmid or integration? | s****n
发帖数: 234 | 3 Microwave method
(Use this for checking integrants)
Smear fresh colony using pipette tip on bottom/walls of a small PCR tube.
Microwave PCR tubes or strips (without lids) for 90 sec in PCR rack.
Resuspend dried out yeast in 25 ul of PCR reaction mix (with Taq already
added) and perform PCR.
Note: Do not overload PCR rack when microwaving--no more than 3 strips
of 8 tubes per microwaving or heat dispersal becomes an issue.
Alkaline lysis method
(Use this for cloning or for checking integrants)
Pick fresh colony using pipette tip into 10 ul of 20 mM NaOH in a 1.5 ml
microfuge tube.
Boil at 95C for 10 min.
Spin down.
Vortex for 10 sec.
Spin down.
Use immediately or transfer supernatant to fresh tube.
Use 1 ul per 50 ul PCR rxn.
Taq PCR to check for integrants
For one 25 ul reaction mix the following:
2.5 ul 10X Taq buffer (NEB Thermo Pol Buffer containing 2mM MgSO4)
0.2mM each dNTP (final conc) (0.5 ul 10mM dNTPs)
1uM each oligo (final conc) (0.25 ul 100uM oligo)
1.5U taq (NEB Taq = 5U/ ul) (0.3 ul)
water to 25 ul
力,
【在 H****s 的大作中提到】
: 各位同学,谁有Yeast Colony PCR protocol分享一下? 现在用的这个太费时间,费精力,
: 并且要好几次才能做出来.谢谢啦.
| H****s
发帖数: 301 | 4 非常感谢这么详细的protocol.这也是我目前也在用的,问题是必须用新鲜的colony,否
则colony PCR成功率就不是很高.
tube.
already
【在 s****n 的大作中提到】
: Microwave method
: (Use this for checking integrants)
: Smear fresh colony using pipette tip on bottom/walls of a small PCR tube.
: Microwave PCR tubes or strips (without lids) for 90 sec in PCR rack.
: Resuspend dried out yeast in 25 ul of PCR reaction mix (with Taq already
: added) and perform PCR.
: Note: Do not overload PCR rack when microwaving--no more than 3 strips
: of 8 tubes per microwaving or heat dispersal becomes an issue.
: Alkaline lysis method
: (Use this for cloning or for checking integrants)
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