How to perform nested PCR? Urgent help plz

[manisha123]

Hi, I am new in lab, and I am unsure how to perform nested pcr, which I have to do for an experiment as my primers are too huge to perform regular pcr.
How is performing nested pcr different from regular pcr? I have 8 different primer pairs to test as it is in 8 different fragments (already ordered primers). Do I perform the pcr test separately 8 different times, and once this is done the dna needs be sequenced. Do I then send these 8 different pcr samples to the lab for sequencing? Please help guys! Thanks

[luisillo]

Nested PCR consists in first amplifying a genome region in a PCR. However , since this region is often too long to be analyzed (electrophoresed, sequenced, etc), a subsequent PCR is performed using the amplified product from the first PCR as template DNA. Considering this, from the different sets of primers, some are used to do the starting PCR and other are use to do the nested PCR. Which means the second pair of primer's sequences have to be present in the DNA fragment that was amplified in the first PCR.

You should check your protocol wisely in order to decide which primer sets to use in the first PCR and which in the nested.

Best of luck.

[manisha123]

Thanks! I do not have protocols. This is a new (small) lab, and I was told to do a literature search to find a protocol specific to that gene. I did not find any so far, so was going to try to use one that i have used for another gene that i did a simple pcr on as that is what my PI told me to do. Do you have different protocols for nested pcr than for simple pcr?
thanks!

[JennYeap]

Nested PCR is almost similar to regular PCR, except the primer and the template are different. For nested PCR, you will use back the PCR product from regular PCR as template, while the nested primers actually falls inside the 1st regular PCR product. PCR protocols are quite standard, for optimization, you can play with the salt concentrations and annealing temperatures.
You have 8 primers for 8 fragments, hence, you should PCR each one separately and the send all 8 fragments out for sequencing to ensure that the amplified products are correct. Two options for sequencing, 1. you can send the purified PCR product straight for sequencing 2. you can clone them and send the plasmid with desired PCR product for sequencing. Obviously the 2nd option is time consuming, but it will be useful when you have other experiments to perform with that plasmid.