Hello fellow researchers,
I am currently encountering significant challenges in a classical cloning procedure and am in need of your expertise. Here is a brief overview of my process:
Image of Gel After Digestion: Image attached
I am also curious why, despite the removal of the insert during vector digestion, there is no apparent reduction in size on the agarose gel. Additionally, running lower concentrations on a new gel seems to still indicate the original size (6500 bp) and the absence of a double band (second image).
Given this context, I am puzzled by the overgrowth of the negative control and the absence of the insert in PCR screenings. Could there be an overlooked factor in the ligation or transformation steps that might explain the high background of vector religation?
I would greatly appreciate any insights or recommendations on potential adjustments to my protocol that could help in achieving successful incorporation of the insert. Could there be additional checks or modifications to ensure the effectiveness of the phosphatase treatment or the ligation efficiency?
Thank you in advance for your assistance!
Some general answers to hopefully help!
You normally will not see a change in size between digested & intact vector. The cloning site has a lot of restriction sites that are very, very close together. The size change is minimal and plasmids don't really run "true to size" on an agarose gel anyway due to coiling.
Expired antibiotics/too low a dose of antibiotics/growing too long at 37*C can make the selection not work as planned.
Ligase is a "fragile" enzyme and easy to accidentally damage if it's not kept on ice at all times. Get a fresh tube.
Can you get a control plasmid like pUC19 to transform into your cells?
I am guessing that one of your restriction enzymes might not be working well. As such, the vector is linearized at one restriction site only (this explains why you don't see a second band cut off from the vector). The efficiency of CIP might be low (this is dependant on your incubation duration), so only a small portion of your vector is prevented from self-ligation. I suggest you check again your restriction enzymes, and elongate the duration of restriction enzymes digestion (depending on the enzyme variants, NEB HF, I usually do 1 hr, non-HF, up to overnight) and CIP treatment (I usually do 1 hr).
1. There is actually no need to treat the vector with alkaline phosphatase after two restriction enzymes, one of which has 5' (like SpeI) and the other 3' (like PstI) sticky ends of DNA after restriction.
2. You need to be sure that the restriction enzymes are working (it looks like one or both of your restriction enzymes are not working. Test them on some plasmid (or lambda phage) where you can clearly see that the desired fragments appear.
another couple of suggestions:
1) If you have enough PCR fragment cut, try to ligate it on itself. If you obtain a ladder of fragment (or a very high MW fragment) it means that the 2 enzymes have cut correctly. If one of the 2 did not work you will see only a band at twice the size of the original one.
2) If you have the same original plasmid that contains another insert between the same RE (SpeI/PstI), you can cut out the fragment and purify the plasmid on a gel, this will ensure that both the RE have worked correctly.
A bit trivial I know, but it had helped me in the past to have a very low background of re-ligation
Here is an update on the recent issues we encountered. We faced two primary challenges: an incomplete digestion process and the cloning strain, which was intended to serve as the host, had already taken up a plasmid. By switching to an alternative strain and using different enzymes, we have successfully resolved these issues.
thank you to all for their support.
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