I have used pet sumo vector, restriction enzymes: bsaI and BamHI, double digestion.
ligation reaction mix : vector (150ng), insert (750ng) total Ligation volume 20 uL
plate 1 : 20ul of ligation mix transformed in 50 μl top ten ecoli cells.
plate 2: 150ng double digested and dephosphorylated vector transformed in 50 μl of ecoli top ten.
(ensuring that nearly same amount of vector is taken in both the plates)
plate 1: 55 colonies.
plate 2: 3 colonies. (control plate)
the difference in the bp of both vector and ligated plasmid of interest is 260 bp.
however, all the colonies show the same size band on the gel as that of the vector.
how is this possible considering the fact that the number of colonies on the control plate was way lesser than the ligation mix plate? (i have screened nearly 20 colonies now)
(no chances of contamination, since everything is being freshly used)
Regarding the ligation, note that the 1:6 ratio should take the insert length into consideration - if your insert is much shorter than the plasmid (probably the case here), you may be adding too much insert.
What are the ligation conditions? are you following the suggested protocol / have a protocol that has worked before?
When you mention that all the colonies give the same band as the vector. Did you extract the plasmids from your colonies or did you perform a colony PCR?
Since the difference in bp between the original vector and the ligated one is relatively small, you may not be able to see it in the gel. Colony PCR would be a better indicator of ligation success.
Did you use a self-ligation control? The control you mentioned (plate 2) only seems to include the digested and dephosphorylated vector. It would be better to prepare a ligation reaction w/o insert having the vector plus ligase buffer and ligase in the right conditions to obtain self-ligation of incompletely digested/dephosphorylated vector. Otherwise you would not get a real estimate of self-ligation background.
I agree that you are probably using too much insert. I use a molar ratio insert/vector around 1/8, but you have to take into account t he size of both of them.
Are you sure that the rests of phosphatase were properly inactivated/removed by purification before ligation? This can be critical.
Regarding the screening the insert is really small. If you do simple digestion to linearize the original plasmid and the plasmids obtained after ligation you would have to run the products for a long time to distinguish subtle migration differences. Perhaps you cant appreciate them.
If you do colony-PCR or plasmid-based PCR with primers flanking the insertion sites of the vector you will probably obtain amplification products of different size depending on whether a given clone is recombinant or not. Alternatively you could do the same with primers annealing at both ends of your insert. A positive PCR would indicate a recombinant clone. This kind of screening is better than just looking plasmid size.
If all the colonies (both control and ligated plasmid) show the same size band, it is highly likely that your transformation was successful, but none of the plasmid vectors successfully carry your insert (ligation failed). Perhaps you need to optimize your ligation protocol.
your insert size is small so its difficult to observe band shifts in uncut plasmids. Do colony PCR of a few colonies and then reconfirm PCR positives using double digestion. However, note that you might have to digest around 1-3 micrograms of DNA for your small inserts to be properly visible as smaller bands incorporate proportionally less EtBr compared to larger inserts and are hence fainter.
Apart from this if it fails then you might have gone wrong somewhere in either your Digestion, dephosphorylation, fragment cleanup or ligation reactions.
Make sure all your workplaces are clean and free from nucleases.
Many times it is not the ligase that has gone bad but rather the 10x ligase buffer as it contains DTT and ATP both of which have a tendency to degrade over repeated freeze thaws.
A good indicator for it is to get a whiff of the buffer. If it's good then it will smell of DTT. Also, after the buffer thaws make sure to mix it properly so that no particulate matter remains at the bottom of the tube.
Gertrudis Rojas I tried to digest the plasmid isolated from the ligation plates.
it is still the empty vector since i dont see any band corresponding to my insert.
I was not aware of molar calculations for vector and insert in ligation mix. I agree i was using too much dna in my ligation mix. (I am relatively new to the clonning process)
I changed the insert vector amount as per the correct formula.
amount of vector* insert size bp* (x for factor by which insert is more)
__________________________________
number of bp in the vector
still nothing works. (1:3, 1:5, 1:10 vector :insert) ratio has been tried. keeping vector always 50ng)
perhaps last think i can think of is to keep ligation mixture probably overnight at low temperature.
or maybe us purified ligation mixture for getting higher efficiency in cloning.
thankyou all for your suggestions though. I wish I hit the jackpot sooner or later.
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