January 28, 2009 at 4:00 am #10812
A PCR product is not inserted, so there is no answer to the question as it stands.
But I suspect that what you want to know is how to make sure that a cloned PCR product has been insered in the right direction in a plasmid. In that case I would do a PCR with one primer on the vector, and the other on the PCR product selected. In that case you will get either a fragment of the size of your choice, or a very long copy of the vector (or very likely nothing if the vector is not quite short).
January 28, 2009 at 8:59 pm #88725pinkypigParticipant
I have heard something about CAP sites or something along those lines? apparantly they are used to add more bases because restriction enzymes don’t like cutting at ends?
Could you explain that in more detail or post link of a website which might explain about CAP sites?
January 28, 2009 at 11:26 pm #88727
Ok what you want is to amplify a sequence and clone it and be sure that it can be inserted in only one direction.
I have never heard of CAP sites, but I know that some restriction enzyme are more efficient when there are overhang (the size depends on the enzyme) before the cutting site. I suspect that what you want is to insert 2 different restriction site in each primer that will make sure that the ligation in the vector (double digested too) can be done in only one direction. In this case my understanding is that you add at the beginning of the primer an extension that contains the restriction site and an extra overhang if necessary.
New england biolabs has a very complete website and usually warn about the need for overhangs for primer design (mostly in the FAQ section of their HF series of enzyme).
I hope this helps
August 1, 2011 at 12:34 pm #105770jayceeParticipant
Read more the next links:
August 1, 2011 at 12:45 pm #105771JackBeanParticipant
and how is that useful? 🙄
October 2, 2011 at 7:55 pm #106558mervParticipant
Firstly, what is the question here? the first post makes very littel sense, the replies I understand.
Hmmmm. Primer design is one answer but you are missing the obvious: RE analysis.
Whether you incorporate cloning overhangs in your primer or not, approx 50% of the clones will be in the direction you wish. So just pick a few of the ones which show the predicted RE pattern, sequence as normal to ensure no PCR errors in the chosen one.
If the question title refers to PCR in one direction, this also is done, but it is an exponential reaction and not a double-exponential reaction, so you have to do a lot more cycles compared to conventional PCR. This is good if you have a conserved region of a gene you can design a primer to but you dont know what the other end of the gene looks like….hence the ‘crippled ‘ PCR – but you can use it to make a probe and pull out a cloned gene say from a library.- you might want to use random hexamers or polyT to the other end…..which may help as a plan ‘B’ (or ‘A’ depending on the reason why you would want to do it!.
October 3, 2011 at 12:53 am #106585
I think someone deleted the post I was answering to….
- You must be logged in to reply to this topic.