How to design random primers - (Aug/24/2005 )
Hello everyone. I am trying to clone an unknown fragment and I figure that it will be better to use random primers. I am new in this and I have two questions about it. 1. Is there any principle in designing random primers? 2. How to determine the PCR protocol when using random primers? Please help me. Thanks.
Wow, there is so much here to say.
Random primers are usually purchased, not bought and used as a primer in a RT rxn. Not a pcr reaction
If I was to design a random primer, I would get a 4-sided die and assign each number the letter of a codon (i.e. 1=A, 2=C, 3=G and 4=T). Then I would roll the dice and record the letter that was shown on the die.
Now, if you are going to use random primers (or perhaps you mean degenerate primers?) You realize that they are going to bind just about everywhere and you will have multiple bands or more probably a smear with brighter bands scattered throughout.
So with that as your product, how are you going to clone out a specific product?
If it is unknown, how are you going to know what size band to cut out of the gel and second, how are you going to know when you have cloned the correct one when it is unknown?
Or, when you fill out the form for the primer design, put NNNNNNNNN