Carried out ligation a couple of times in different ratios and incubation times. Transformation results: negative has more colonies than positive.
Really appreciate the help, thanks.
No it is not difficult to ligate 300bp into a vector.
But, if you can provide more details like the enzymes you are using, insert to vector ratios and whether your insert is a PCR product and the vector you are trying to clone into it would be helpful to narrow down why you haven't been successful.
I also found it sometimes tricky to clone 30 bp parts via restriction cloning in vectors. Here some possibilities to overcome that problem:
- using PCR to just add on that small region on your primer, then PCR the backbone with Restriction site on both sites -> cut - ligate - transform (dont forget to Dpn1 digest the template tho)
- Gibson assembly worked vert efficient for me on small parts like that. I recommend 50 bp overhangs, 25 on the insert and 25 on the backbone for each site.
Good luck!
Hi Ht Liang,
yes sometimes cloning very short DNA fragments can be surprisingly hard. A little more information would be useful. What is the source of the fragment? What ends are you using? What are the numbers you are seeing?
You say that you have a reduction in numbers with the insert, did you check to see if they had the insert in them? If I see a change in numbers over the background, be it up or down, it's telling me something is happening - I've had correct inserts from reduction in numbers before. Now it could be that only one of your ends is ligating, so that you don't get any inserts but have reduced the number of self ligating vector molecules. If your background is high then you also have a problem with generation of the vector backbone - more information is needed to determine if this is so.
-Richard
Hi, first of all make sure to avoid insert overkill. Calculate numbers of molecules and mix the ligation in a reasonable ratio. The number of insert molecules should not exceed the number of vector molecules more than by factor of 10. You can make use of this online DNA calculator:
http://www.molbiotools.com/dnacalculator.html
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques