protocol cloning/expression

Use this category for questions regarding problems manipulating proteins in molecular biology applications (expression, detection, etc.)

Moderators: mdfenko, leekaming

protocol cloning/expression

Postby xtnw58 » Sep 16 2016 8:57 am

Hi,
I have a list of plasmid constructs from a previous student working in a different lab. They are currently in pACYCDuet-1 vectors. My supervisor told me to transform them in Top10 cells, miniprep them and over expressed them in BL21 for protein purification.

Are the transformation- mini prep steps only necessary to create a stock here in the new lab?

In this case the protocol should be quite straightforward:

- transformation in Top10 cells, make plate
- pick a colony from plate and make O/N culture
- use MINIPREP kit on the O/N culture

And now I'd be ready to transform in BL21 cells for over-expression

Am In forgetting anything?
Why for the first step are we using top10 cells? I know they are good for cloning but I am only making stocks..

Thanks in advance,
Xile Yu
xtnw58
newcomer
newcomer
 
Posts: 4
Joined: Sep 16 2016 8:32 am

Re: protocol cloning/expression

Postby relaxin » Sep 19 2016 1:17 pm

Transformation of the plasmid into TOP10 cells is for a stock (for safe keeping) and for purification of more plasmid DNA. With the plasmid DNA, you can transform BL21 cells for protein expression.

For protein expression, it is better to make fresh transformation into BL21 cells. The foreign protein may be detrimental to the bacteria. So it is not safe to keep the transformed BL21 cells as stock.
Retired academic researcher. Mention of a specific product does not imply my endorsement of the product. No conflict of interest or guarantee to work on the advice given. Do as I say, not as I do. Not liable to the loss of your valuable samples.
relaxin
PI of Posters
PI of Posters
 
Posts: 7165
Joined: Jan 11 2006 12:40 pm
Location: Mauna Kea

Re: protocol cloning/expression

Postby xtnw58 » Sep 22 2016 10:44 am

Thanks,
it's what I thought.

Now I have to send the Miniprep samples of the pACYDuet plasmids for DNA sequencing. Which primers should I tell the company to use? And why?

Thanks,
Xile
xtnw58
newcomer
newcomer
 
Posts: 4
Joined: Sep 16 2016 8:32 am

Re: protocol cloning/expression

Postby relaxin » Sep 23 2016 11:10 am

You can use AcycDuelUP1 and T7 terminator Primers. For primer sequences, see the vector map pdf below. T7 promoter cannot be used, since there are two T7 promoters in the vector for the expression of two genes.

http://biochem.web.utah.edu/hill/links/pACYCDuet-1.pdf
Retired academic researcher. Mention of a specific product does not imply my endorsement of the product. No conflict of interest or guarantee to work on the advice given. Do as I say, not as I do. Not liable to the loss of your valuable samples.
relaxin
PI of Posters
PI of Posters
 
Posts: 7165
Joined: Jan 11 2006 12:40 pm
Location: Mauna Kea


Return to Protein Methods

Who is online

Users browsing this forum: No registered users and 1 guest