Dear Kamel,

I suggest that is to re-PCR the DNA product or, you can start purification kits are usually reduce the concentration of DNA at the end. So another way you can think about is to 1X Phenol-chlorofom cleanup.

My best Regards,

Muhammed

You can take a fraction of weak PCR band product and reamplify the same.Then you go for the purification followed by  Restrction digestion.

all the best 

Simply you can go for 2round of PCR with your 1st round pcr product 2-5microlitre as a template for second round PCR, so that you will get enormous thick band in the gel. To visualize in the gel it needs 20ng DNA concentration, so your 1st round product is below that, if you do 2 round you will get more than sufficient amount of DNA concentration.

Yes, you can (but hopefully this weak band is really the band you want).

1. Do a gel-extraction using a kit (such as Qiagen Gel-Extraction kit), re-do a PCR. If you don't have a kit yet, store the excised gel block in a freezer.

2. Then, run a gel to check the quality and size, and finally sequence it to verify its sequence before you do downstream experiments

Good luck.

You already have got few suggestions which all would help you. I would like to add that you can run multiple PCR reactions or just scale up your reaction to give you larger amount of the desired product or try to increase the number of cycles by 5, if it helps. If you perform multiple reactions, pool the final product and then go for PCR purification protocol using kit.

good luck

Navjyoti

Thanks all for your suggestions. I also want to ask, cant I purify the product from 1st reaction and Do another PCR rxn with it?

No need to purify your 1st round product, if you do so, u will loos further dna concentration. so better you can go directly do 2 round from 1st round product and then seen in the gel and then purify before going for next step.

Hi, generally purification decrease the yield. so in your case if you have just one specific band you can make purification by exosap ... it is really good and do not decrease the purification yield and give us good results, it is just enzymatic purification

dear Kamel. in most of the cases the purification wid kits decreases the final purified product. and i think there is also a problem in 2nd round PCR with the same primer pair. Some times you can't have any product at all during 2nd round. so my suggestion is that you either do multiple pcr reactions then combine them n purify them or use more initial template (instead of 1 or 0.2ul, 2-3 ul) or make an initial preamplification (5-10 pcr cycles) of larger fragments than your  target fragment  and then use this product as a template for your actual pcr reaction.

hoping this might help you. Best of luck