I see 2 Problems in your strategy:

The main thing is that your PCR product is not getting cleaved properly. You put your restriction sites directly at the 5' end of the primer. Restriction enzymes need some additional basepairs for efficient cleaving (https://www.neb.com/en/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments?srsltid=AfmBOopjAvp9kudTfzexRpcpeWfsHBeWcj0ryKAjIIYDKiWitP6DC5Vc). I always add at least 4 random basepairs to my primers to avoid this issue.

The second thing is that you are using a too high ratio between vector:insert. In general, the closer the vector and insert are in size, the lower the ratio should be. In your case I would try to use a 1:3 molar ratio for ligation. Also, in my experience overnight ligation at 16 °C works better then 2 h at room temperature.

Hope this helps!

Hi Khadijah,

I completly agree with Todor Tschongov that you should add additional bases to your primers at the 5' prime end (4-5 bp) to improve cleavage and to try another molar ration . 1:3 sound good to me as well as the overnight ligation.

However I have checked your cloning strategy - When I blasted the sequence you provided I found the L protein of the Nipah virus but the annoted sequences diverged from your provided sequences on the DNA level, nevertheless the protein sequence was the same. That you have another DNA-Sequence might depend on the strain - the only problem I see here is that, if you are not totally sure that you have the sequence of the strain you are working with you could get difficulties with the primers.

However the primer sequendes you provided are fitting to the provided sequence.

Nevertheless - I think I spotted a major issue with your reverse primer - it seems that you want to add a His-Tag to you protein - correct?

You also added a stop codon.

The problem is that this is your 3' prime end primer (reverse primer) and your gene specific part as well as the stop codon are correctly in reverse complementary orientation - however, it seems that your His-Tag sequence is not! The His-Tag sequence in your primer is the sequence of the coding strand. With this primer you will get the following protein sequence "IIGYISIIVVMVMM* instead of the sequence I think you would like to get (IIGYISIIHHHHHH*). If you want to have the His-Tag the sequence of your reverse primer has to be :

ctcgagtcagtgatggtgatggtgatgGATGATGCTGATGTAACCGATGAT or

ctcgagtcaGTGGTGATGGTGATGATGTGATGATGCTGATGTAACCGATGAT

(both sequences should work)

In addition you should add some bases to the 5' prime end (as already mentioned).

I would strongly recommend to double check this - maybe I got something wrong but I must admit that I don't think so.

Regarding your fw primer - there is additional bp between the restriction site and the ATG you included - is there any reason for this. It might not disturb but I also think it is not necessary and add also some base pairs at the 5' prime end to improve cleavage

.

I am not totally sure if I get what you did with the pUC/M13 primers? Did you try to do PCR on pfastBAC? I have looked for several pUC/M13 primers available and none of them is annealing to pfastBac. But maybe I got something wrong or looked for the wrong primer sequences. If I got it correctly you should consider using other primers

Nevertheless, your sequencing results from the plasmids isolated from the BL1 cell indicated that you cloned at least something. Colonies of different sizes might indicate that your gene product is toxic for the cells. in this case the growth of the cells with the correct plasmid could be inhibited leading to smaller coloies. What also could happen is that you get only colonies with mutated sequences which are less toxic for the cells.

So if you do the cloning again (with the correct reverse primer) you can try to grow the colonies at lower temperature (20-30°C) or use a cell line which is designed for coping with toxic gene products (if you are still expierence problems).

If you are interested I can send you an ApE-File with the sequence you want to clone (with the His-Tag) inserted in the correct position between the restriction sites. In ApE ( I don't know if you know the program) you can also search for seqeunces (eg for the sequences of primers you want to know and look up restriction sites).

I know this was a lot but I hope this will help with your cloning strategy.