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
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
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?