HI michael - please see my thoughts below:

"I am working on cloning a 3 kb gene into pET-47b using engineered restriction sites. Sequencing showed that my gene was successfully ligated into pET47 but it contains 7 missense mutations. I sequenced plasmid from a 2nd colony and it again had 7 missense mutations but they were at different locations. I am new to cloning and I was wondering if this is a common problem? So far I have 3 guesses of what the problem could be:

1) UV light while gel purifying the PCR product digest (I think I tried to keep the UV exposure to a minimum. I have also not directly exposed my desired lane to UV in the past, instead I used a small amount in a separate lane which I can use to determine the position of the band. I do not remember what method I used for this band)."

People are obsessed with gel purification - I never understand this since it's a lot easier to use a PCR cleanup column - anything that can be used to clear primers from a PCR product will get rid of your digested ends.

"2) Old/bad polymerase used in the PCR amplification. I am using the KAPA2G Robust kit which I understand to have very high fidelity but it is close to 2 years old."

Two years old? Hmm. I am never (read, "never") a fan of people dipping in and out of one another's reagents. Get your own kit - preferably a new one. KAPA produce a different kit for "long range" PCR, from the description of the one you're using, it seems this kit is more designed for qPCR.

"3) Would a high concentration of dNTPs in the PCR reaction cause missense mutations? I did increase the concentration of dNTPs in the PCR reaction (used 1 uL) because my gene is 3 kb."

so - 1uL isn't a concentration, it's a volume. Sorry to be pedantic, but it's important you are clear about what you are doing. That being said, in principle, high dNTP concentrations might lead to errors, but it's unlikely, and if you're using a proof reading enzyme or enzyme cocktail it ought not be.

"Are any of these problems a likely culprit?"

My solution is - get a new PCR kit, designed for cloning longer fragments (3kb isn't so long really) redo the reaction with the recommended concentration of dNTPs (i.e., don't 'mess" with it), and sequence 20, not 2 clones. Despite what you might be led to believe from reading papers, it doesn't always work first time!

Good Luck

G

Your problem comes from PCR polymerase enzyme with poor proofreading function. Try New england Biolab's Phusion High Fidelity enzyme for your PCR amplification. For smaller DNA fragment PFU enzyme would have been fine but 3kb is way too long. I have used both enzymes but I like Phusion better for its speed and consistency. By the way, if you are lighting UV light underneath of your gel on a glass or plastic plate, UV exposure should not be a problem. Good luck!

HI michael - please see my thoughts below:

"I am working on cloning a 3 kb gene into pET-47b using engineered restriction sites. Sequencing showed that my gene was successfully ligated into pET47 but it contains 7 missense mutations. I sequenced plasmid from a 2nd colony and it again had 7 missense mutations but they were at different locations. I am new to cloning and I was wondering if this is a common problem? So far I have 3 guesses of what the problem could be:

1) UV light while gel purifying the PCR product digest (I think I tried to keep the UV exposure to a minimum. I have also not directly exposed my desired lane to UV in the past, instead I used a small amount in a separate lane which I can use to determine the position of the band. I do not remember what method I used for this band)."

People are obsessed with gel purification - I never understand this since it's a lot easier to use a PCR cleanup column - anything that can be used to clear primers from a PCR product will get rid of your digested ends.

"2) Old/bad polymerase used in the PCR amplification. I am using the KAPA2G Robust kit which I understand to have very high fidelity but it is close to 2 years old."

Two years old? Hmm. I am never (read, "never") a fan of people dipping in and out of one another's reagents. Get your own kit - preferably a new one. KAPA produce a different kit for "long range" PCR, from the description of the one you're using, it seems this kit is more designed for qPCR.

"3) Would a high concentration of dNTPs in the PCR reaction cause missense mutations? I did increase the concentration of dNTPs in the PCR reaction (used 1 uL) because my gene is 3 kb."

so - 1uL isn't a concentration, it's a volume. Sorry to be pedantic, but it's important you are clear about what you are doing. That being said, in principle, high dNTP concentrations might lead to errors, but it's unlikely, and if you're using a proof reading enzyme or enzyme cocktail it ought not be.

"Are any of these problems a likely culprit?"

My solution is - get a new PCR kit, designed for cloning longer fragments (3kb isn't so long really) redo the reaction with the recommended concentration of dNTPs (i.e., don't 'mess" with it), and sequence 20, not 2 clones. Despite what you might be led to believe from reading papers, it doesn't always work first time!

Good Luck

G

Thanks, Shamol and Grant!

I will try a different polymerase.

Grant - I first tried the process without gel purification and my final product was a clone with a truncated insert. When I did the gel purification, there was a second band under 1 kb. I missed this when I did an analytical gel the first time. Now I do a column clean up after PCR and then the gel after digestion.

The Kit I am using is mine but I have it in our lab because we were using this to sequence some previous patient samples. I read the specifications for the kit and it seemed like a 3 kb was in range. I should have just ordered a more appropriate kit though.

I'm sorry for not giving a concentration of dNTPs. I was asking this question from home so I did not have the numbers in front of me. I think we would usually use 0.2-0.5 uL of the dNTP mix so I should have said that it was 2-5x concentrated. The kit suggested using a higher concentration for larger amplicons so I do not think that this would cause the type of problem I am experiencing.

Thanks again for your help!

Dear Michael

I go with Grant concerning the purification from the gel. Just digest your PCR fragment and purify it over a column. Even if you have several bands, don´t bother. After ligation we screen our clones with PCR using external primers to find clones with the right length (if you use one external and one of the starting primers you can even check the orientation in the same step) and use this fragment directly for sequencing. And like Grant we always sequence several clones in parallel.

Good luck

Michael

I see this is rather a DNA proofreading problem than anything else. But, if people would love to spend extra time and steps to fix a simple problem, I say more power to them. And, at the very end, a scientist would not even figure out where the problem really lies. I am always for simple fix for a simple problem. Thank you.

Hi Shamol - of course, pragmatically, what you say is correct, get the clone however and move on, but don't you think that there is value in *understanding* the processes that we all use in kits and whatnot, to prevent us revisiting the same problems again and again, in slightly different guises?

Maybe being older and less lab active, I have more time to reflect on such things, rather than be faced with the daily necessity of getting to the end by the fastest means.

Very best

G

Hi Grant, Thank you for your response. Actually, I am not a proponent of using any kit without understanding the fundamentals. I was only in favor of changing one single critical variable and that would be the enzyme- provided everything else is optimally added to the reaction. You are very correct that people are using kits without knowing how things really work. And, that is very sad. Yes, your answer is very detailed and complete. Thank you.

UPDATE: New polymerase worked great.  Cloned the gene for three enzymes using the same method.  Thanks everyone.

Great News! THanks for updating us. GOod luck with the rest of your work

best

G