I am a PhD student trying to identify Aeromonas and Plesiomonas shigelloides with pcr using housekeeping genes, gyrB and hugA respectively. I am currently using the heat lysis method for dna extraction from pure isolates. I pcr tends to have amplified multiple regions and hence been having multiple band sizes for most isolates. I am lost as to why this is happening. Can anyone who have had similar experience or have knowledge as to the possible reason kindly provide guidance. Thanks in advance.
hi
1- run the PCR first time by positive and negative control to optimize PCR condition ( such as MgCl2 concentration , annealing temperature ,...)
2- after bacterial heat lysis , you should centrifuge at 13000 RPM 10min in room temperature for cell debris sediment .dilute DNA by TE buffer 1:5 to 1:20( for multiple band sample )
3- you can use DNA extraction kit To get clean DNA with high purity and a certain concentration.( for Equal amount of bacterial DNA template )
http://www.epibio.com/docs/default-source/protocols/quickextract-bacterial-dna-extraction-kit.pdf?sfvrsn=8
good luck
Hi,
I agree with Reza Hadavi. You should also check your primers against you DNA sequence. Your primers may be annealing at multiple regions in the sequences where they are complementary. Optimizing the reaction will also help with this. Question, have you considered using primers for the 16s RNA gene or the 23s RNA gene region for bacterial identification?
I agree with Reza and Maria. Please check your centrifugation rate, you may increase it to 15000RPM. Check the primer sequence too, subject it to BLAST on NCBI to be sure. If you continue to get same multiple bands, consider using another primer that has been mentioned to be specific for your organism in literature.
Also looking at your ladder on the gel picture, it seems the PCR condition is not optimum. You may need to optimize with positive and negative control only first so as to be sure the primer is okay.
Thanks all for your response. I did optimise for both positive and negative controls and it seems to work fine for them. The first and fourth bands after the first ladder is my positive control. And the second and third are negative controls. I have also ran a primer blast as suggested the primers seem to be quite specific. Here is an image of subsequent run for some positive samples and some showing multiple bands. I optimised the pcr condition by reducing the annealing and extension time. Thanks once again
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