I have performed RFLP for WT and mutant sample. The WT sample was only PCR product, while mutant band was extracted from agarose gel electrophoresis (it was checked on gel again to see the integrity of the purified band). When the restriction digestion was performed, the wild type sample was digested perfectly while there was no band in Mutant sample not even the undigested band. There was even no amplified band. What could be the possible reason? does the enzyme digested all the DNA??
Concentration of gel is so important based on your product lengths after digesting by the enzyme.
There is always loss of material purifying from agarose gels and for digestion you need enough dna to see all of the bands . If you have 4 cut sites then 5 smaller fragments will take up less ETBR and may not be vivible. Now that you know that the enzyme cuts in the presence of pcr buffer it is simplist to cut both the normal and the mutant samples directly from the pcr reaction. You can always run a checker gel to see that the product has cut
If there is no band, that could mean the PCR product of the mutant was not extracted from the gel properly. Because, even if the enzyme did not digest the mutant, the uncut DNA still would show band after RFLP. If the enzyme digest all the DNA, there should be bands of different lengths, or at least, a smear of DNA.
Dear Humaira Naeemi,
If the problem, occurs for the first time then you doubt on your restriction enzyme. 1.You can precheck whether your enzyme has the ability to cleave the target or not. 2. It is better to maintain duplicates to avoid a lot of confusion. 3. While retrieving you must ensure your kit or chemicals are free from inhibitors, after all they show bands when you recheck with possibly less intensity because of bits and pieces of DNA. 4. Another chance could be your diluting your DNA at the first place while retreiving from agarose gel, you might loose your target of your interest. Which is not abundent to make the enzyme to detect and digest your target site.
Conclusion - Recheck, Eliminate inhibitors, Always use direct products for the crucial experiments like RFLP , Check the enzyme applicability to your target.
I hope this information will provide you the right understanding to your problem. All the best for your future endeavors.
Cheers,
Srinivas
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