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Digestion necessary after PCR? - (Mar/17/2014 )

Hey!!! I have a problem I thought I could get help with here.

 

I have a gene of interest I wish to insert into a new vector.

The recipient vector has already been cleave/digested, and electrophoresis showed it was successful (2 separata bands). Now this sample is stored in -20C until I can ligate the gene of interest into it.

 

Now, I am having difficulties with the gene of interest!!!! I miniprepp DNA, measure the concentration, and run a PCR with primers that flank the gene of interest. This PCR should give me amplified amounts of the gene of interest, right?

Gene of interest has recognition site X on 5'-end and Y on 3'-end.

Recipient vector has recognition site Z on 5'-end and Y on 3'-end.

But the sites X and Y are generated by enzymes that have COMPATIBLE ENDS. So now I am assuming that I can purify the PCR-sample (get rid of primers, dNTPS, salt etc) and just ligate it with the recipient vector (which is shares compatible ends with).

 

However I am being told that I haft to digest the PCR-product BEFORE the ligation. And I don't understand why?

Isn't the PCR-product constituting of the gene of interest free from the vector, and ready to be ligated into the recipient vector?

 

Also, even when I do the digestion, I run a electrophoresis afterward and that only gives me 1 single band. How should I interpret that?

 

Please help!!!!Appreciate all the help I can get.

Thanks

-Biologystudent-

You want to do a digestion of your PCR product for a couple of different reasons. First, if you are using a Taq family polymerase it will add an extra A on your PCR product. This A will hinder any ligation even if your PCR product has compatible ends. Second, what do you mean when you say your PCR product has compatible ends? Are you trying to do a blunt ligation or did the vector digestion produce overhangs? You should complete a digestion regardless. When you are setting up your ligation, try using both your digested product and your undigested PCR product for insertion into your vector. Hopefully that will help clarify some things. good luck!

-jerryshelly1-

You wrote "This A will hinder any ligation even if your PCR product has compatible ends".... but..... I WANT to ligate in the end process. So isn't that counterproductive?

 

 

I am using the Phusion polymerase.

With compatible ends, I mean that the 5' end of the insert will be compatible with the recipient vector it will be ligated into.

The 3' end of the insert/PCR product and the 3' end of the recipient vector are cut with the same restriction-enzyme.

However, the 5' end of the insert/PCR product and the 5' end of the recipient vector are cut with DIFFERENT restriction-enzymes, but they will still create sequences that will match together for the ligation.

 

Another question.....

I am told that

- if you have a sample from a mini prep (no PCR done after mini prep), then you can digest the sample and verify the digestion with electrophoresis (successful digestion gives 2 separate bands)

 

- but if it is a PCR-product (miniprepp and then done a PCR with respect to the gene of interest), then I can't validate the digestion with electrophoresis because it will still give 1 single band. Why????

I haft to do PCR ----> PCR purification ------> digest -----> ligate -----> and NOW I can run a electrophoresis to validate the experiment.

So, basically, I understand why I can do the electrophoresis in the last step..... but I don't understand why I can't do a electrophoresis after the digestion-protocol????

 

 

You want to do a digestion of your PCR product for a couple of different reasons. First, if you are using a Taq family polymerase it will add an extra A on your PCR product. This A will hinder any ligation even if your PCR product has compatible ends. Second, what do you mean when you say your PCR product has compatible ends? Are you trying to do a blunt ligation or did the vector digestion produce overhangs? You should complete a digestion regardless. When you are setting up your ligation, try using both your digested product and your undigested PCR product for insertion into your vector. Hopefully that will help clarify some things. good luck!

-Biologystudent-

Your wording is a little confusing so if I don't answer your question right let me know...

 

1)- if you have a sample from a mini prep (no PCR done after mini prep), then you can digest the sample and verify the digestion with electrophoresis (successful digestion gives 2 separate bands)

 
Do you mean that you can verify the presence of your PCR in your vector post ligation via digestion? Yes you can do this and it will give you two dropouts. 
 
2) - but if it is a PCR-product (miniprepp and then done a PCR with respect to the gene of interest), then I can't validate the digestion with electrophoresis because it will still give 1 single band. Why????
 
I am assuming you are using two primers that have the RE designed on the 5' ends? You will only see one band because an agarose gel can not resolve a 2-10nt product that you will be digesting off the end of your PCR product. 5'-NNNN-RE Site-Gene of interest-RE Site-NNNNN-3'.
 
 
Does that make sense?

-jerryshelly1-

Thanks man!

 

Sorry if I don't make sens in my writing. English isn't my first language.

What do you mean with "give you 2 dropouts"? I am assuming its the same meaning as "giving you 2 bands" with respect to the electrophoresis.

 

 

So, I think I got it.

1) A mini-prepp sample that has not been PCR-amplified afterwards, CAN BE subjected for the digestion-protocol and followed by electrophoresis in order to deduce whether the digestion was successful or not. 1 bad indicates that the digestion was not successful, but 2 bands indicates the digestion was successful.

 

2) However, a sample from a min-prepp that has been PCR-amplified afterwards,..... well then it is useless to run a electrophoresis because due to the amplification I cannot obtain the resolution necessary to distinguish between the digested end-products (2-10nt long) & the the PCR-product itself.

 

 

3) Baiscally today I will then be doing the following...  let me know if the procedure is correct:

 

a) Miniprepp DNA-sample from the transformed bacteria that I inoculated yesterday

 

b) Measure the DNA-concentration from mini-prep using a nano-drop

 

c) run a PCR with primers designed to flank my gene of interest

 

d) run a electrophoresis to validate that the PCR was successful... I want 1 crisp clean band of the right size that I am expecting

 

e) if electrophoresis validates my PCR, then I can go on and purify the PCR-product

 

f) I will now digest the PCR-product, This step is necessary to create the complementary sequences at the 5' and 3' that is necessary for the subsequent ligation with the recipient vector

 

g) Ligate this digested PCR-product together with the recipient vector

 

h) finally, NOW I can run a electrophoresis to find out whether the whole digestion, ligation and experiment was succesful

 

Correct?

 

Previously I used to run a electrophoresis AFTER THE DIGESTION (after step f), and get 1 single band. And interpret that as that my digestion was unsuccessful.

So, if I am right.. this step (electrophoresis after step f) was completely unnecessary since I wouldn't be able to distinguish whether the digestion was successful or not. Correct?

 

Sorry for the long post and detailed description (being detailed and list everything in order helps me to avoid mistakes and understand better)

 

 

Your wording is a little confusing so if I don't answer your question right let me know...

 

1)- if you have a sample from a mini prep (no PCR done after mini prep), then you can digest the sample and verify the digestion with electrophoresis (successful digestion gives 2 separate bands)

 
Do you mean that you can verify the presence of your PCR in your vector post ligation via digestion? Yes you can do this and it will give you two dropouts. 
 
2) - but if it is a PCR-product (miniprepp and then done a PCR with respect to the gene of interest), then I can't validate the digestion with electrophoresis because it will still give 1 single band. Why????
 
I am assuming you are using two primers that have the RE designed on the 5' ends? You will only see one band because an agarose gel can not resolve a 2-10nt product that you will be digesting off the end of your PCR product. 5'-NNNN-RE Site-Gene of interest-RE Site-NNNNN-3'.
 
 
Does that make sense?

 

-Biologystudent-

Everything you said was correct. Good luck!

-jerryshelly1-

Thanks man!

 

I got a follow-up question now.

After step g (see above) where I ligated the vector and insert, I did a normal transformation protocol to get the complete vector now into competent bacteria and let them grow overnight.

Today I will pick up 1 colony and grow that overnight again in LB+antibiotic medium.

Tomorrow I will do a miniprepp and run a electrophoresis.

 

But I wonder.... can I set up my experiment as following:

Step 1) Run a 1st electrophoresis after the mini-prepp.

- If successful you should see 1 single band indicating that the insert has been taken up by the vector and it is intact

- If not successful you should see 2 separate bands (vector and insert)

 

 

Step 2) Then I can cleave the vector with REs that I will know where they cleave (preferably RE that cleve outside the insert so it will be left intact) and yield pieces of DNA of determined sizes. This cleaved sample, I can run a 2nd electrohproesis of to determine the outcome of the cleaved products, in comparison with the ladder.

And the electrophoresis form step 1 can be compared with the electrophoresis from step 2, as a quality control and reassurance.

 

Would you say it is a good setup, or will it suffice with just doing:

mini-prepp----> cleavage protocol ------> 1 single run of electrophoresis?

 

 

Everything you said was correct. Good luck!

-Biologystudent-

You can check it that way as well, the only thing is that your final ligation volume may be too small for you to successfully see a dropout.

-jerryshelly1-

hi everybody! i'm new one.

actually i'm doing PCR-RFLP to test Ace-1 mutation in anopheles

and after the amplification i digest the pcr product with Alu I enzyme.

 

after migration, the undigest pcr productt shows 541bp (normal), but when it's the digest pcr product i get some bands but not all (i get 403bp, and i should get 253 and 138bp too but the latter don't appear ???? and  the migration is not flat, like i have some laces on gel! i don't know why? i've repeated it several times with performing my mastermix but  no good results.

 

 

sorry for my english, but i need help!

 

thanks

-andt-

You could help us understand better if you provided detailed information about your PCR and digestion methods. Especially the digestion and gel preparation, starting after the PCR reaction. I would suggest (if it is easy) to sequence a few of your PCR reactions to make sure the restriction sites you expect are really there.

-phage434-