The substrate used for activity assay are L DNA 40 mer R DNA 30 mer complementary to T DNA 80 mer.. these substrate after incubation at desired temperature are electrophoresed through urea denaturing 15 % polyacrylamide gel
The expected bands after ligation is 70 bp size other than 30, 40 and 80 mer but there is an unknown band of 50 bp why??
This band appear in the negative control without enzymes by mixing three substrates .
In the picture attached below the first two lane after ladder showing ligation reaction and 3rd lane is negative control.
In negative control the top band is 80 and there is no 70 bp band that are present in ligation sample below 80.
The last two band is 30 and 40 respectively
But why i am getting a band of 50 mer ??
The 50 bp band is likely due to a self-ligation reaction between the two 30 mer primers. When the primers are complementary to each other, they can form a hairpin loop structure that can be ligated by the DNA ligase. This results in a 50 bp product that is half the size of the expected 70 bp product.
It can be prevented by using primers that are not complementary to each other, using a higher concentration of DNA ligase, or using a DNA ligase that has been modified to prevent it from ligating to self-complementary sequences.
Thank you @john odonnel for you valuble suggestions.
But the unknown band appears even in a negative contol without DNA ligase when only primers are mixed .
The lane three in the picture is negative contol without DNA ligase , so there is no ligation un the negative contol that's why the 70bp band does not appears but the unknown band is still there??
check each primer individually to see if one has a 50bp sub-product (could be a failure in the chemical synthesis of the 80 mer)
the problem can be related to the used ligase which may not have thoroughly ligated to the mentioned substrates and it leads to formation of both expected 70bp product and the unexpected 50bp band.
If the problem is not related to the ligase, it could be due to a secondary structure formed by the DNA substrate.
@maryam Baniasadimoosaabadi Thank you for your Value suggestions..
The band appears when 40 mer and 80 mer are mixed even without Ligase so the problem is not with ligase .
Kindly would you like to tell me how can i check that either the substrates making secondary structure or not??
@Dadier Poncet Thank you so much for your reply..
I have individually checked all three primers and the band didn't appeared in any of them..
The band appears when 40 and 80 mer are mixed or when all three are mixed..?
Below i have attached the gel.
After ladder the 1st band shows 30 mer then 40, 80, 30+40, 80+30, 80+40 and 80+30+40 in last lane
@maryam Baniasadimoosaabadi
If any of the the substrate are making secondary structure then the band should appeared when they are run individually?
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