My experiment involved ligation of DNA size 800 bp (which was amplified from ligated DNA sized 400 bp each) with DNA size 400bp to create fragment of size 1200bp. I did electrophoresis of the ligase mixture to see whether the ligation worked, and there is partly ligated strands and non ligated strands. When i do PCR from this ligase mixture, all three strands are amplified equally, which is problematic since i need specific amplification of the DNA fragment size 1200bp.
I changed the amount of cycles, and amount of DNA used as template, and annealing temperature but I still obtain amplified fragments of all three fragments.
Is it possible that I can put m ligase mix in gel electrophoresis and cut out the required fragment (1100bp) and use it as template for PCR? Or is it possible that i would obtain the same result nevertheless?
Or is it better to do PCR from my ligase mix, and cut out PCR product from the gel and use that as template?
Thanks in advance for your advice :)
Dear Anakha Satish
It is always recommended to gel elute the ligation mixture or restriction digestion mixture, if it is incomplete (in case of yours), so in the following steps the possibility to obtain non-specific product will be reduced. You can cut out the desired fragment from the gel after ligation and use the clean and purified DNA as a PCR template. If your PCR reaction contains non-specific binding, lower bands will appear which you can determine by checking primers specificity. If higher bands appear, decrease the extension time of PCR.
All the best.
You can purify the band but for a quick result just poke a pipette tip into the band ( preferably from a LMP agarose gel) then dip the tip into a pcr mix and cycle
It is generally not recommended to use ligase mix directly as a template for PCR, as it may contain contaminants that can interfere with the PCR reaction. It is also possible that the partly ligated strands and non-ligated strands in your ligase mixture may interfere with the PCR reaction and lead to the amplification of all three fragments instead of just the desired 1200 bp fragment.
Cutting the gel to isolate the desired fragment (1100 bp) and using it as a template for PCR can be an option, but you should take care to minimize the potential contamination during gel excision and purification. Additionally, you may want to run a negative control reaction using the gel slice without any DNA to ensure that the gel extraction process does not introduce any contaminants that could interfere with the PCR reaction.
Alternatively, you could try using PCR primers that are specific for the 1200 bp fragment and adjust the PCR conditions to optimize amplification of only the desired fragment. If you continue to get non-specific amplification, you may want to consider redesigning your primers to increase their specificity or consider using a different approach for generating the desired fragment.
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
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