Okay, I have been constructing a plasmid shuttle cloning vector for a certain bacterium. First, I inserted the plasmid DNA from my original source and pUC19, then I put erythromycin gene. Now, I am trying to clone a 300 bp promoter region with XhoI at 3' end and SalI at 5' end (artificially added after PCR). However, I don't seem to get good transformants. I've been stuck for months now. It's either I have a plasmid DNA which is too low molecular size or a plasmid without the promoter insert. What am I doing wrong? Please help me.
I use E. coli DH5a strains and the plasmid vector now is about 7 kb in size.
You may be having some sort of procedural issue. Do you perform a double digest of the plasmid and PCR product (both enzymes at the same time)? If so, are you certain the buffers for both enzymes are compatible? What do you use to purify the cut plasmid and cut PCR product? Have you double-checked that your primer sequences are correct for the restriction enzymes on the PCR product? Is your PCR product the correct size? How much plasmid versus how much PCR product are you including in your ligation reaction? What are your stopping points during this process? How do you store reactants during stopping points?
Thanks for your response.
1. First, i tried doing double digest at the same time in plasmid vector and insert gene (after PCR), both enzymes at the same time. But it seems that double digestion didn't work well, because I got transformants in my self-ligation vector (as a control).
So after trying multiple double digest (both at the same time) and failed many times, I tried to do stepwise single digests and purification/gel extraction steps in between. Then ligation. But still false positives shows up.
2. Buffers seem compatible for both enzymes, I use Takara enzymes and buffers and I specifically designed the restriction enzyme sites so that the buffers are compatible.
3. I use Macherey-Nagel PCR clean up gel extraction/purification kit. I used gel extraction after double digestion, then when I did stepwise single digests I perform gel extraction after the second restriction enzyme was used. So XhoI digest > pcr clean up > SalI digest > gel extraction > ligation (i also tried switching the order of enzymes)
4. PCR product is correct size and sequence is correct. I tried two option, digest pcr products directly and clone pcr products to TA vector and then digest and purify the insert gene afterwards. I also checked the sequence reqlgion through sequencing to check the accuracy of the sequence. All looks well in the sequence.
5. I tried using different molar concentrations of insert:vector such as 1:3, 1:5, 1:10.
4. I don't have much stopping points usually. But when I do, it is when the digestions are complete. Between restriction digestion and ligation, but not normally.
When you are checking your product, have you tried using different restriction enzymes? I have had problems with certain enzymes on occasion and had to change to a different set to check my products. Also, when you do the initial restriction digests of your PCR product, do you maximize the amount of PCR product in your restriction digest (no water in the reaction)?
1. Yes, I use other enzymes too several times already. Some are cut but no insert gene, and some plasmid DNA were just mutants
2. I don't do ligation from pcr prodduct. I tried it before, but I don't get any transformants. I changed my method to clone PCR products to TA vector first, then check proper sequence, then I cut and gel extract the insert gene from there.
You may be having a problem due to the quantity of your insert. Gel extractions lead to extreme loss of product, and in my experience, you need significantly more of your insert sequence in a ligation reaction for successful insertion.
@Breanna_Symmes: Okay. Usually I get 20-50 ng/ul after gel extraction of insert sequence. Then I used insert:vector molar concentration of 3:1, 5:1 and 10:1. Do you suggest I increase more?
@Sulman_Shafeeq: Yes, they are unique restriction enzyme sites because I added them to the vector before this step that I am clueless and having problems with. I also confirmed by digesting them singly in agarose gel electrophoresis.
Hi,
XhoI and SalI are compatible so you need to to a CIP + phenol extraction.of your plasmid.
An other way (for the next time) is to ligate XhoI of the plasmid with the SalI of your vector and the same in the other side. You do the ligation and then you digest this ligation with one of the enzyme. Don't forget to inactivate the enzyme before to transforme your bacteria and plate all the transformation.
Eric
thank you @Eric_Flatter. i get the first comment, but can you clarify the 2nd paragraph. i am confusedwith the "vector" and "plasmid", aren't they the same?
Sorry ! I do a mistake !
The idea is to do a ligation with destructions of the restriction sites and use a digestion of your ligation with this enzyme to do an enrichissment. So :
"An other way (for the next time) is to ligate XhoI of the plasmid with the SalI of your insert and the same in the other side. "
Interesting method. Looks complicated. Have you tried this method? Sorry, I am so new to this work. But thanks for your suggestion. Can you walk me through the principle of your protocol, please?
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