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- Most Active In Molecular Biology (535 posts)
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- Age 64 years old
- Birthday January 25, 1948
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Cambridge, MA USA
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mycoplasma genetics and metabolism
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In Topic: What temp the lid of the thermocycler must be set?
Yesterday, 06:06 PM
Most cyclers with heated lids will automatically set the lid temperature, slightly above the block temperature, or above the highest block temperature if things are cycling. If you need to set it for a 50C reaction, you might set it at 55C.
In Topic: Why do supposedly sharp bands sometimes appear unclear in agarose gel?
24 May 2012 - 05:04 PM
You told us absolutely nothing about this gel.
In Topic: A faster way to pick single colony clones for PCR screening?
23 May 2012 - 05:11 PM
I do it by setting up a plate or strip with 50 ul pure water in each well. Then I use a 10 ul tip on a pipettor to touch a colony. The tip is suspended in the water and scraped against the sides of the well. The same tip is used to deposit 3 ul of the water onto a gridded master plate, then discarded. Afer picking colonies, a multi-channel pipet is used to transfer 0.5 ul of the water into a 10 ul pcr reaction with one primer on the insert and the other on the vector backbone. The master plate is incubated and the correct colonies prepared from the colonies with successful PCRs. If you feel confident, you can grow up some 10 ml cultures from a small number of the picked colonies on the chance that one of them is correct, potentially saving some time.
In Topic: Size Bias in Minipreps - Which method is the best?
23 May 2012 - 05:04 PM
Rather than relying on not having size bias, why not make the plasmid sizes identical except for a small bar code difference? Then you would not need to worry.
In Topic: Ethanol precipitation - bad step
23 May 2012 - 04:10 AM
It's almost certainly recoverable. What are you doing with it next? You could purify the plasmid by essentially doing an ethanol precipitation. Add 1/10 volume of 5M NaCl, 2.5 volumes ethanol, spin down hard and look for the precipitate. Wash with 70% ethanol, and finally redissolve in TE. If you started with salts in your initial solution, or if you added sufficient ethanol, you could omit some of these steps.
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