I am trying to generate stable cell line (HEK293T) by using the X- plasmid. I got negative result by using the intact (circular) plasmid so this time I am trying to use linearized one. The problem is that, after cutting and gel purification, the cut-plasmid yield is very low (15-35 ng/ul) and I need high amount of plasmid (1ug) in multiple wells. I was just wondering that can I use cut plasmid without gel purification after phenol/chloroform extraction and ethanol precipitation (as the heat inactivation is not applicable to my enzyme BspT1 cat# FD0834)?
Secondly, I don't have PCR fragment purification kit. Can I use the gel extraction kit for purifying my cut DNA (12kb) in liquid form instead of gel? Thank you.
If your yield is low after gel purification, you should possbly rather check your restriction and purification, since they don't seem to work very well then. Do you dephosphorylate the cut plasmid? You could possibly also use two enzymes generating overhangs to cut, to reduce religation even further.
However, the concentration you discribe is also not necessaily small, depending on how much you put in the restriction and in how much solvent you redissolved the DNA. From the values you've given I can't see if you only put in 100 ng into the restriction mix for example.
However to answer your actual questions:
Some kind of purificatio would be advisable, to get your salts, enzymes etc. out. It does not necessaryl have to be a gel purification. A Liquid DNA purification Kit with a binding column could possibly work just as well.
If your particular kit works should be easy to find out, since there is a manual (either in paper form or online) which normally tells you, for which kb size range the column is suitable. Also there are often additional information available online (including some user modified protocols) which could help you to decide if the particular kit is suitable for your purpose.
Purification of PCR product by gel extraction kit should work fine. I used to do it in some cases with the E.Z.N.A gel extraction kit from Omega Bio-Tek, but I don't see why it shouldn't work with other kits as well.
Yes, you can use the linear form directly without an intermediate gel purification step. Also, it is OK to use the gel purification kit to purify DNA from a solution, although depending on the kit, you might have to adjust the concentration of the binding buffer. In most cases the manufacturer will have some modified protocol in their website for this purpose.
Beware that the quality of DNA preparations from silica-based columns is not great for transfecting DNA into mammalian cells. You would be better off using an ion exchange-based kit for this case.
Hope it helps!
In the past I had great success using miniprep kit. I used the miniprep kit to isolate the plasmid DNA from bacteria and then regenerated the column so that after linearizing my isolated plasmid for cloning I could just run it through the column again. Of course it uses size exclusion so you have to make sure the size corresponds, but it isolated the linearized DNA very well and cleaned it up nicely. I always had decent yield and it was high transfection quality DNA. I used this to get super clean linearized backbone for cloning.
I always did gel extraction for my PCR products and it worked great. You can also use columns to clean up PCR products (dependent of size), they will flow through and column will catch any enzyme, salts or backbone you want to avoid. If you need a protocol for this I might have one lying around somewhere.
Hope this helps!
Thank You Cori for sharing your experience! I will try your strategy too.
- Badges
- Science method