It's difficult to suggest without knowing more. How different was the sequence? Are we talking missing nucleotides or mutations? Is it the same changes that occur with both cloning attempts. Are the errors inside the region annealing to primer? Can you still see restriction sites - assuming you cloned sticky ends that have ligated to compatible ends in a vector. Just to confirm that the digest worked and ligated to vector correctly.

The most common errors are sequence errors in the primer, restriction sites too near the end, missing an internal restriction enzyme target site, taq with high 5'-3' exonuclease activity chewing away the primer ends. Miss-priming of the primer in first round of PCR if 3' end of primer is only designed to anneal to a very short region of your gene. You could redesign primer to anneal to at least 21 base then add your restriction site, then add 5 bases.

Dr. Amanda,

Kindly have a look on the data obtained (attached).

There are both missing and mutated NTs. However, few clones perfectly matched in 5’. They are, however, not the same changes occurred. These changes are within coding sequence (oligo flanked). My restriction enzymes are working as per my observation in cutting vector within 1 h. I was trying to insert the target within EcoRI/XbaI digested stick ends.

Length of the oligo is 35 bp, excludes the miss-priming possibility. Can you guess anything more closely?

Hi Navaneethaiyer,

I've taken a look at your cloning and I don't see anything wrong with your plan but your sequencing is indeed very troublesome.

To address the single site mutations throughout the clone, are you using a high fidelity polymerase such as pfu and keeping cycle number small, say around 25-30 max?

Have you looked at the sequencing profile to check if it is just a base-calling problem with the sequencing reading software or is it a real mutation?

As for the the 3' end errors, they do appear to be quite random, I can't see any logic there at all. If you want to keep the primers you have, I would follow the suggestion from Andrei above to create a blunt end at the end of your PCR for cloning.

Aside from this, are you aware that XbaI cannot digest methylated DNA? Your PCR product is OK, but your vector will need to be passed through a dam- bacterial strain before isolation to prevent methylation.

Good luck and keep us posted if there is anything else we can help with.

Amanda,

as for XbaI the methylation interference depends on the flanking sequence. Usually, XbaI sites in MCS are not affected by dam.

see the link for the interfering sequences:

https://www.neb.com/faqs/1/01/01/is-xbai-affected-by-methylation

(no personnal interest with NEB company ....)

Hi

You may confronting with a mutation hot spot at 3’-end sequence proximal to stop codon. you may use a mixture of Pfu, Taq (like Maxtaq-vivantis, etc.), reduce PCR cycles to 30. Moreover your primers may don' have spacer sequence (for restriction enzyme sitting), so it is better to clone the PCR product into TA vector, sequence it with vector universal primers like M13 and then subclone it into desired vector.

God speed you

Hi Navaneeth,

It seems like you are sequencing only from one end of the clone. I am surprised that you are getting very good reads beyond 1000 bases. Usually, the quality of reads start to fall off after about 800 bases. Have you tried sequencing from the 3' end of the clone? Typically for a sequence this long it is advisable to sequence from both ends and overlap the two. I bet you that you will get good clean reads. I don't think that there is any issue with the cloned fragment itself. The few point mutations may be PCR artifacts or sequencing artifacts appearing beyond the 1100 to 1200 bases from one end. If you do the reverse sequencing you will get clean reads in this region without mutation. Sometimes, individual specific SNPs are likely to be present. I typically sequence 6-10 clones to identify one clone without sequence deviations.

Another thing to consider is potential errors in the PCR primers themselves. This is a common problem that is missed when people sequence with the same primers used to generate the PCR product.