Designing Primers For Cloning From Pure Fungal Cultures

Question

[H@rry]

Hi,

I am new in cloning and likewise interested to clone 1 gene from fungal DNA (Mycelium). Now the problem is I designed primers from the complete CDS sequence of gene with 516 bp length. My primers forward is with start codon ATG but reverse with not stop codon as it is in the 3/4th part of gene. The amplicon size of gene is 389 bp but I am able to amplify the full CDS fragment ~ 516 bp on 1 % agarose gel. I have few queries regarding this as follows.

1. If it good idea to proceed in this case with such primer for cloning, as the getting size is equal to I am expecting.
2. Since, I designed the primers from comleate CDS and they are without RE (both forward and reverse) does it make some difference. If yes, would it be good idea to add REs now to the primers or I need to design new primers from genomic region.
3. Here are my primer sequences are 5'- ATGTGGCATATCTCGAAGTAC-3' and 5'-TGGCATATCTCGAAGTACTGA -3' (without reverse completment)

Thank you in advance

[Damian Kao]

This really depends on what you want to use this clone for later and what vectors you are using. And like dpryan said, it's not really a bioinformatics question. I suggest http://biology.stackexchange.com/

[Devon Ryan]

1. Does the gene not have 5' UTR? Usually you want to clone that too as it'll affect expression.

2. "RE" means restriction enzyme or something else? It's rather difficult to follow what you wrote. Whether to add restriction enzyme cut sites depends on the vector you want to clone things into (some use a traditional MCS (multiple cloning site) that needs restriction digestion followed by ligation, others just need a single A overhang).

BTW, your question has nothing to do with bioinformatics.

[H@rry]

Thank you for your answer

1. I have only CDS sequences of gene.
2. Yes RE means Restriction enzymes. Sure, I will use MCS too in my case.

Answer 1

[Devon Ryan]

1. You should always sequence a clone afterward, so yeah, go ahead if the PCR product is the expected size (and you don't have multiple bands).
2. If you need to cut the target vector with a restriction enzyme to get it cloned into it (e.g., you're not TOPO cloning or similar), then you should add them onto your primers. Make sure to add enough overhang for the enzymes to work (NEB has a datasheet on that).

In the future, you'll probably get faster/better feedback on a biology-specific forum, since this doesn't actually have anything to do with bioinformatics (though a number of us come from biology or other backgrounds).