Which method for DNA purification b4 cloning - (Apr/04/2014 )

I need to digest a plasmid with NotI, dephosphorylate it, clone it to another plasmid, and finally do the heat-shock transformation.

 

One step-wise procedure is:

 

1. NotI digest

2. Run a small amount on agarose gel to confirm digestion

3. PCR purification

4. Do the Alkaline phosphatase reaction

5. Run the whole reaction on agarose gel

6. Separate the linearise, dephosphorylated DNA by gel extraction

7. DNA cloning

8. Transformation (heat-shock method)

 

I've been thinking that since I will have to do step 2 (above) why not run the whole NotI digest on gel and separate linearised DNA. This way I will only have my required linearised DNA available for alkaline phosphatase to work on. Also, I will avoid step 6 (above) and only have to do PCR purification instead. Here's what I was thinking:

 

1. NotI digest

2. Run the whole digest on agarose gel

3. Separate the required linearised DNA by gel extraction

4. Do the alkaline phosphatase reaction

5. PCR purification

6. DNA cloning

7. Transformation (heat-shock method)

 

My question is::

 

1. Isn't second procedure more convient and quick?

I would skip step 2 entirely - just do the dephos in the digest then clean up and run on a gel to extract the fragment.  Most of the time dephosphorylation can be done directly in the digestion mix with the addition of  the dephos buffer.

 

Note that dephosphorylation is usually only done on the target vector - you shouldn't need to do it for the insert and it may be easier to amplify the insert rather than subclone.

I would do this:

 

1. NotI digest

2. Dephosphorylate using shrimp alkaline phosphatase in the same buffer (might be able to do the digestion and dephosphorylation at the same time). Check the requirement for zinc in some phosphatase reactions -- you may need to supplement the buffer.

3. Heat kill (won't work for CIP)

4. Use small amounts directly in a ligation reaction.

 

No need for gels except to check for digestion. No need for PCR cleanup. The less you do to your DNA, the fewer things can go wrong, and the more product you will have. Design your protocols to avoid extra purification steps, and (in my opinion) especially gel purifications.

 

Do not dephosphorylate your insert! Your ligations will not work if both vector and insert are dephosphorylated.

Thanks for your replies.

 

If I don't do gel extraction after NotI digest then in subsequent ligation reactions there'll be five possibilities:

 

1. re-circularisation of insert and vector. --> Vector will produce colony

2. insert-vector ligation (the one I want). --> Will see colony

3. insert re-ligates to its own digested fragment. Same happens to vector. --> Again vector will produce colony

4. instead of insert its digested out fragment ligates into vector. Same happens to vector. --> Vector will produce colony

5. no ligation whatsoever. --> No colony

 

If I do gel extraction of inset and vector after NotI digestion then I can be sure that the colony will either be re-circularised vector or insert-vector. Thus, my chances of picking the right colony increases.

 

I've decided to not do dephospho reaction.

 

The new plan is:

 

Digest vector and insert with NotI (can we digest vector and insert with NotI in same reaction?)

Gel extraction of NotI-digested insert and NotI-digested vector.

Ligation reaction.

Heat inactivation.

Transformation.

 

What you guys think?

The heat inactivation is unnecessary. I would expect a LOT of background with this method. You will also see both orientations of your insert. The major challenge will be finding the (relatively few) clones which actually have your insert.