we have extracted dna by ctab method from tender leaves
this is for 10 microliter reaction
why did we get multiple bands for rbcl?
Kindly provide suggestions to improve my work by viewing the attached file
Thank you all.
I agree with the above and would add that you have another problem and that is that all of your amplifications have a primer dimer band below 100bp. This is caused by the pri,ers annealing together and they amplify very well so your proper amplimer then amplifies less well. You may want to think about using a hot start enzyme, less primer or addibg one of the important parts of the reaction ( enzyme or magnesium or primer while the reaction mix is held at 70c then immediately start cycling, This should give you more product and the higher annealing temperature will give you a cleaner product
Hello Leelavathy,
Getting multiple bands during PCR amplification may be due to unspecific binding of the primers. You can minimise it by increasing the annealing temperature of your primers to increase the primer binding specificity.
Hello Leelavathy,
I also suggest increasing the PRIMERs length until 25bp if they are too short in order to improve its specificity.
I agree with the above and would add that you have another problem and that is that all of your amplifications have a primer dimer band below 100bp. This is caused by the pri,ers annealing together and they amplify very well so your proper amplimer then amplifies less well. You may want to think about using a hot start enzyme, less primer or addibg one of the important parts of the reaction ( enzyme or magnesium or primer while the reaction mix is held at 70c then immediately start cycling, This should give you more product and the higher annealing temperature will give you a cleaner product
- I basically agree with Paul
- your best bet is to use a hot start Taq principle or a hot start highly progressive polymerase particularly NEB phusion: the advantage of such an enzyme is that you can amplify at the actual Tm with high efficiency
- This results in highly efficient clean PCR products
- Avoid if you can reducing Mg especially if you increase the annealing temp as that can lead to less efficient PCR and insufficient product
1) Excellent read for PCR reactions set up: http://genome.cshlp.org/content/3/3/S18.full.pdf
For you, especially the section "ANALYZING PCR RESULTS ". Good luck.
2) https://www.neb.com/tools-and-resources/usage-guidelines/guidelines-for-pcr-optimization-with-taq-dna-polymerase
- Badges
- Science method