Hi all,
I have recently been attempting to add a C-terminal 6xhis tag to my gene of interest. My reverse primer for this consisted of ~20bp homology to the end of the ORF, omitting the STOP codon - 6Xhis tag - XhoI site - Leader sequence. I successfully amplified the gene and subcloned into pJET 1.2. I have screened the clones by PCR and by restriction digest which indicated the gene of interest was successfully cloned. When sequencing, the end of the gene is correct, however, the tag has not been incoorporated and the STOP codon has remained. My assumption is that during the initial PCR, the gene has amplified correctly but the polymerase has not read through to the 6his tag etc. Since tagging primers are so large the Tm's for the whole primer are very high, thus I calculated by Tm based on the 20bp of homology to the target gene. Any help/advice would be much appreciated. Please ask if any other information would be helpful!
Dear David
you can add the Histag directly in your plasmid by PIPE cloning trhough performing a vector PCR with primers containing flanking regions those overlapping and contain the histidine tag sequence.
In the following links
https://proteocool.blogspot.com/2021/09/tips-of-week12-rapid.html
Article Combining the polymerase incomplete primer extension method ...
you can find some more informations about it.
good luck
Manuele
Hi David,
I think you have proceeded with the right approach. I have done a similar procedure in my lab for incorporating a His-Tag. However, I find it extremely unlikely that the STOP codon would be amplified as well.
I would advise you to pay close attention to the template DNA used for PCR. A common mistake would be to use a plasmid containing the gene of interest, bearing the same restriction sites and the AmpR marker, this would lead to false positives in both colony PCR and RD. Using gel-purified PCR product for pJET cloning or amplifying the GOI from a different source would avoid these false positives.
Hope this helps, do let us know how it turns out.
Good luck
Try increasing the extension time during the PCR so it has time to go to the end. You can do an initial cycle at the lower temperature and then do the remaining cycles at the higher temperature for more stringency. Do you have a stop codon after your 6xHis tag?
To add a C-terminal 6xHis tag to genes of interest using PCR, you can follow these general steps:
1. Design gene-specific primers: Design PCR primers that include the desired 6xHis tag sequence followed by a stop codon and any necessary linker sequence. The forward primer should bind to the 5' end of the gene of interest, and the reverse primer should bind to the 3' end of the gene.
2. Amplify the gene: Perform PCR using the designed primers and a DNA template containing the gene of interest. Use a high-fidelity DNA polymerase to ensure accurate and reliable amplification.
3. Purify the PCR product: Purify the PCR product using a DNA purification kit. This step removes any unincorporated primers, nucleotides, and contaminants from the PCR reaction.
4. Prepare the expression vector: Choose an appropriate expression vector that allows for easy cloning and expression of your gene of interest. Linearize the vector using restriction enzymes, taking care to cut just after the stop codon of the gene within the vector.
Ananth Krishna Narayanan Robert Adolf Brinzer
Hi Ananth and Robert,
Thankyou for your reponse. I have attempted the 6his tagging following your recommendations and I am increbily happy to say that the modifications worked! For reference I used a gel extracted product and modified the PCR program to progressively increase the anneal temp over time! Thankyou again for your help!!
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