3 replies to this topic

[siddharthsameer]

Hello all
I did the mini prep of my sequence which is 1.4 kb and the Maxi prep of my vector (pPICZalpha A) which is 3.6 kb.. I  followed the complete protocol ... Now What would be the next step to verify my midi and maxi prep..Since I am doing all this stuff for the first time and that to alone in the lab, I seek some help and advice from you...
kindly give me a quick response.... I would be thankful to all.
regards

[bob1]

Verification can be done by sequencing or by restriction digest.

Note that miniprep usually has a specific context for molecular biology - it means a small scale preparation of plasmid DNA, not an insert alone.

[klenow]

Hi,
As bob1 says, miniprep refers to a purification of plasmid DNA... not inserts or digested plasmids. After electrophoresis I use to check the correct size of both insert and digested vector (use a low-power UV lamp...) and then cut the correct bands. After that, I use a kit specifically designed to purify double strand DNA (from agarose or from PCR reactions... ). I usually check the DNA concentration and then I perform the ligation. For that I usually try 1:3 to 1:10 molar ratios (digested plasmid:insert). You can find ligation and transformation protocols in the forum. Then, you have to see if the ligation worked and select some colonies. I like to confirm that the colonies have the proper insert in the proper vector by doing colony-PCR (basically, you amplify a region containing your insert and some part of the vector). From the positive clones, I culture some of them, do a miniprep, keep a glycerol stock and sequence them.

Edited by klenow, 20 August 2012 - 01:50 AM.

[LabLackey]

Alternatively, you could find a restriction digest strategy that would also indicate whether the vectors (after ligation) have the insert.  Our lab usually suggests a restriction site unique to the insert, although you don't necessarily have to use it.