Currently, I am using Q5® Site-Directed Mutagenesis Kit for introduction of desire mutation in genome. instead of getting single desire band, I am getting multiple band on agarose gel;. I have cross verified DNA template, Primers.
DEar Rohini Nangare
to help you we need to have more information about the sample that you load, the plasmid size and the marker that you use.
i suppose that the most intense band is low size than the band that you expect from the vector amplification (that have to be more or less ). I'm right?
if Yes, it seem that you have some non-specific primer annealing.
You can try 2 different solution:
1) Screen different primer annealing temperatures
2) Use a different DNA polymerase
Generally i perform the mutagenesis using the PIPE cloning approach, which is quite similar to the Q5 sire directly mutagenesis kit in principle, and i prefer
Kapa Hifi (Roche) or Clone AMp (Takara) polimerases to the Q5 which in my hand work very well for smaller dna fragments but is less efficent for amplify the entire plasmid.
if you are interested you can find some more information about PIPE cloning on my blog PRoteocool (https://proteocool.blogspot.com/)
good luck
Manuele
Hi Rohini Nangare ,
I will try my best to help you with the limited information given. I have used the Q5 Site-Directed Mutagenesis kit extensively throughout my Master's and PhD, and for me, it has worked with troubleshooting at times. I have had the issue amplification of other DNA bands in the past which were non-specific or undesired. In the event of such situations, I would go back to the beginning and modify the ratio of primer and DNA added to the reaction mix. Trial and error with the annealing temperature is also another approach that should be investigated. Usually, when the manual suggests the time lengths for amplification in the last step based on the size of the DNA, it should be followed to the point. When I have shortened the time length in the last step in the past, I have seen amplification of undesired DNA as well. I hope this helps and good luck with the SDM!
@Manuele Martinelli
Thank you sir for your response.
Actually top most faint band is desired band, others are nonspecific.
I'll give you details very soon.
Meera J. Patel
Hello,
Thank you for your response.
I also modified the ratio of primers, template DNA and followed the manual instruction but still not getting desired PCR product.
Rohini Nangare ,
Hmm...check to see if there may be contamination in any of the starting materials - primers, DNA template, buffers, etc. All components in the reaction mixture are significant including the time durations and if anything is incorrect, it will result in undesired results. SDM and PCR are very sensitive techniques. Good luck!
Meera J. Patel
Hi,
I am getting nearly expected PCR product on agarose gel. After screening of colonies, not getting plasmid with desired mutation.
Is there any suggestion you can give me ?
Hello Rohini Nangare
How many colonies are you screening? It depends on the length of your mutation and where it is located in your plasmid. Since my plasmid was very long, I used to screen about 40-50 colonies and would get a few potential positive clones. After doing further workup with PCR confirmation, I would be able to narrow down to maybe 3-4 plasmids that have the desired mutation. Further DNA sequencing would then confirm this. SDM is a very tedious and sometime laborious process, but once you have checked off all the verifications, you are ready to go. Good luck!
Meera J. Patel
I have screened almost 20-30 colonies. Size of my plasmid also more than 10 Kb. and mutation inserted in middle of the plasmid.
From your opinion, I need to screen more colonies to get desire clone. Right?
Rohini Nangare
Yes, I would try to screen more colonies. Are you doing blue-white colony screening? That might help to pick out the positive clones as well.
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