Hello everyone,
I am trying create fluorescent tagged versions of two proteins, but unfortunately I cannot get my cloning to work, although I have changed a lot of things it's still not working.
So I have designed primers for amplifying the genes out of the plasmids they were previously cloned in. I have introduced restriction enzyme sites within my primers, and I added 3 nucleotides before the restriction site. My PCR works fine and I get nice strong bands. Also I have tested and optimised the restriction designs by single digesting my plasmids (I use enzymes from NEB). After optimising the times all enzymes gave nice single bands.
After the PCR reaction I gel clean my PCR products using the Qiagene gel clean kit. Following that I double digest my PCR products and my plasmids (the plasmids were perched from add gene) and I run the products on a gel and I gel clean the double digested PCR products and the double digested plasmids. Following the gel clean I de-phosphorylate the plasmids using CIP from NEB and I gel clean once again.
My ligations were set at different vector to insert ratios starting from 3:1, 1:1, 1:3, 1:5 and 1:7. I have set the ligations at room temperature for 1 hour or overnight at 16 degrees. For the ligations I used the T4 Ligase from NEB and I set the ligations according to the NEB protocol.
For my transformations I have used DH5a E.coli, and 10-beta competent E.coli from NEB. For the transformation I have used the full volume of my ligation (20 μl) to 50 μl of competent cells. I kept the cells on ice for 30 minutes and then I heat socked the bacteria at 42 degrees for 1 minute, and then they were placed again on ice. for 5 minutes. Following that I added 300 μl of LB or SOC to the cells and I incubated the transformed bacteria at 37 degrees on a shaking incubation. I plated the transformed bacteria on selection plates and incubated overnight at 37. After having set 60 ligation reactions I only got one colony on one plate which proven to be just a self ligated plasmid.
I really do not know what else I can change or how I can make this to work. I feel that the problem is found in the PCR double digestion, but I do not have a way to test that.
Any ideas or suggestions will be highly appreciated.
Thank you in advance.
Hi Olga,
1. Most likely your phosphotase is still active. How did you kill it? In general phosphotases are very stable and difficult to kill, but CIP is extremely stable. I would recommend you to switch to Shrim phosphotase and do treatment before gel cleaning.
2. Cutting PCR products is very inefficient especially if restirction sites are just at the ends of fragment. 3 bp might not be enough.
Dear Olga,
your explanation is long but some info are missing.
Your positive transformation control (uncut plasmid) is working?
Your positive ligase control is working (single cut plasmid+ligase or double cut plasmid+control insert+ligase)?
How much DNA are you using? Do not exced 50 ng plasmid, more is bad, if using ultracompetent cells use much less (eg. only 2 microliters of ligase reaction).
There are still some possibilities, but need to know controls results
- check your PCR products by restriction (choose a restriction site that does not lie at the borders of the product). If they are processed correctly, you should have the right products
- check your ligates on an agarose gel (you should observe a decrease in fragment intensity and the appearance of multiple bands that indicate ligated DNA fragments)
- perform a transformation with a reliable plasmid and calculate the transformation efficiency of your bacteria
- don't use more than 5 µl of ligate in your transformation protocol
I hope these ideas can be of help to you.
Greetings, Christian
Hi Olga,
All the above is good advice.
The one thing I saw in your method is adding 20ul of ligation to 50ul cells, old wisdom is no to exceed 1/10 volume of ligation to cells.
Another thing to try is to clone the PCR product into a TA vector such as pGEM-T easy or pCR™2.1. You will have to have used taq polymerase to at least add an extra A at the end of the reaction. Doing this will make it certain that the digestions on your insert fragment has worked before you cloning into the final volume.
-Richard
You've got some great advice above.
When things go wrong with cloning, start doing all those controls that you're supposed to do, but that we usually leave out when things are going well.
Transformation and ligation controls: Cut you vector with a single enzyme, purify it, then set up two ligation reactions, one with and one without ligase. Set up three transformations:
a) uncut vector
b) ligation reaction - ligase
c) ligation reaction + ligase
You should get a load of colonies on (a), any problems here are problems with you competent cells. You should get no colonies on (b), if you do then your digestion isn't working well enough. Self-ligation should be very efficient so you should get quite a few colonies on (c). If you don't, then there's either a problem with your ligation itself, or it's not transforming efficiently.
This should pinpoint where your problem lies, and you can investigate in more detail. Make sure these steps are working really well before trying to go any further. If you only get a few colonies in (c), then really optimise your ligation and transformation first.
My view of what your main problems are likely to be:
1. You're adding too much ligation reaction to your transformation. 20 ul added to 50 ul competent cells is way too much. I generally add 1-2 ul, and as mentioned above don't go over 1/10 vol. Less is often more with cloning, adding too much often decreases the efficiency. Likewise with your ligation, I tend to use 50 ng vector in a 10 ul ligation reaction, don't go much over that.
2. Alkaline phosphatase still active, so preventing ligation. As mentioned above, shrimp alkaline phosphatase is better.
3. Gel purification. I know many people gel purify everything (as you do in this protocol) and clone fine, but it really reduces ligation efficiency in my hands. I try to avoid it wherever possible. In this case, if you've got a single band with your PCR, then don't bother gel purifiying, just do a PCR cleanup with a spin column. Similarly with your vector, if the bit you're removing is
Hello,
thank you so much about your answers. They are all very useful and I will follow your advice to switch to Shrimp Alkaline phosphatase instead of CIP and see if I get any results. Also I will run the controls and see how these work.
Thank you so much, and I will keep you up to date.
Kind regards
Olga
What do your concentrations look like after the gel extraction? I just recently got my cloning to work and I'm so excited! I wrote up all of my tips/ things that worked here: https://www.theweekendtraverse.com/
Nicole Golden, thank you for the recommendations!
I read your block and congratulations for your cloning working!!!
I only got one of the two genes working. I think something was wrong with my primers for the second one, but unfortunately I am done in the lab, so future students can try it!!!
Good luck with the rest of your experiments!!!
Hey Olga Biskou,
I have also been trying to clone a 3.1kb gene into pET47b(+) and pET 28(a) vector using primers designed with restriction sites (SacII, XhoI & NdeI , XhoI respectively). I have put ligation reaction in different molar cones. 1:2, 1:3, 1:5 and 1:7 and have been getting colonies but they always come out to be false positive.
Can you please help me out and give suggestions if you were able to solve your cloning problem.
Thanking You,
Deepika
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