Question

i honestly have no idea how to answer this one. can someone please help me out with a thorough answer so that i can learn from it as well.

You have a forward primer that anneals within sticky fingers and a reverse primer that anneals within the Pen-tag (see pictures below). Xba1 EcoR1 Xho1 Sal1 Pst1 Tbd1 ReversePen-tag Forward primer 40 Sticky fingers e) We used blue white selection to figure out which colonies of E.col had our insert in lab. This time our expression vector does not insert into the LacZ gene. How can we use PCR on each colony that we pick to determine which colonies have sticky fingers inserted? Please explain. lonies of E.coli had our insert in lab. This time our

Answer 1

If you have to select which colonies have the vector with the sticky fingers sequence cloned in it,

First you have to pick colonies and extract dna .

Then , you will use this dna as template for doing PCR

( polymerase chain reaction) Through the use of this tecnique it is possible to detect and amplify an specific dna fragment.

For this purpose, it is necessary to use 2 primers that are complementary to the ends of the fragment to be amplified

In this case, to detect the vector that have the insert of sticky fingers sequence, you have to use 2 primers

A - a forward primer that is indicated in the figure, that will recognize and binds to a dna sequence of sticky fingers fragment

B- a reverse primer, like the one in the graph, that will bind in a region present downstream the site where the sticky fingers fragment was inserted ( if you see in the vector exists a polylinker region where there are several restriction sites sequences .This is site where the cloned fragment is inserted,)

So if the sticky fingers fragment is in the vector, you will obtain a pcr product because, the forward primer will bind to its complementary region in the cloned fragment

If this fragment is not present in the vector, the forward primer will cannot bind and no amplification occurs .

So, in this case, if there is no amplification product, you can conclude that sticky fingers is not inserted in the vector.