probe size for DIG-labeled - (May/01/2006 )
I have problem to search fragment containing my small target fragment. It is only 90 bp!
At the moment, I have this small fragment cloned in pGEM-T vector.
Then I will use this fragment as a probe for southern hybridization with digested genomic DNA.
The problem is the size of probe is very small and there are 2 ways to prepare probe;
- If I cut plasmid, I will get fragment size 108 bp.
- If I amplfy by PCR using M13 primer, I will get fragment size 360 which most part of fragment come from vector.
Is there somebody has ever used very small probe with DIG system?
Please give me some advice.
Or, 2 choices of my probes, is it possible to get the target fragment?
The easiest approach is to order two primers for the ends of your insert, and PCR out the probe. This will be easier than cutting it from the vector, and will be more selective than the M13 PCR. I would guess that the M13 PCR probe would work, however, depending on the complexity of the genomic sample you are blotting. The PCR approach has the benefit that you can do the PCR in the presence of DIG labelled dUTP (Roche) to label the probe at the same time you are PCRing it.
Thank you very much for suggestion!
By the way, probe size will be only 90 bp. Will it work well?