I have following gel as my Colony PCR result. I see that there is a band between 100 -200 bp. But there is a large amount of DNA in some wells like one in 11 which doesn't migrate. I have seen this happening a lot with my samples.
What is happening?
I am using 6X Safe-white safe view DNA stain buffer to stain my samples.
I think that the non moving band in 11 is genomic dna or protein. The pcr has not worked in the majority of samples and you are getting a lot of primer dimer. Since your last 2 samples worked we can say that the pd is from the reaction not from contamination so you should consider that as the pcr is working well it may be that the cloning failed and that these samples are negative for the insert
Dear Amar
To answer your question, please take a look at the links below.
https://www.google.com/amp/s/www.researchgate.net/post/What_are_these_bright_PCR_products_that_dont_migrate_through_the_gel/amp
https://www.google.com/amp/s/www.researchgate.net/post/What_could_be_the_cause_of_PCR_product_bands_not_moving_from_the_well_of_the_gel_electrophoresis/amp
https://www.google.com/amp/s/www.researchgate.net/post/PCR_products_stick_in_gel_wells_PCR_problem/amp
https://www.google.com/amp/s/www.researchgate.net/post/Why_PCR_product_is_sometimes_intactnot_run_in_wells_of_Agarose_gel_and_not_run_over_the_gel_though_the_gel_and_electric_supply_is_alright/amp
- Colony PCR in my experience is hit and miss
- If your DNA is bound to protein that that can lead to it becoming stuck in wells as you see
I recommend you to purify your DNA stock, then try to check the specificity of the primers and good luck
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