Hi Monika

Primers are synthesized chemically so all the primers may not be complete and of same length. Desalting is done to remove the chemicals used during chemical synthesis. PAGE /FPLC/HPCL purification ensures that primers are uniform and of desired length, incomplete primers are removed. In  desalted also more than 90-95% primers are compete. I have performed many site directed mutagensis with desalted primers and it has worked. May be you try to change PCR condition for desired amplification. If you provide details of PCR reaction mixture and conditions some suggestions can be given.

If you are not obtaining mutations it sounds more like you are recovering template so your DpnI treatment has not been effective rather than any failing of the oligo quality. Try extending the DpnI treatment or adding a little more enzyme. After that a column clean-up of your product should be enough, if you want to gel purify avoid or minimize UV exposure, try blue light and a 'safe' stain.

We use this method for producing mutations all the time and have never used purified oligos. As Mrityunjay says even desalted primers are generally  still reasonably pure but this purity decreases with oligo length as the addition of each base is not 100% efficient so you end up with a pool of mixed length primers but the majority is good. Unless you are using very long primers (>50-mer) to introduce more than one mutation in the same step desalted primers should be more than adequate. The worst case scenario is that you may have to pick a few more clones.

One thing to keep in mind is that this is still an amplification step so the fidelity of your polymerase enzyme is key. It is rare but we have occasionally found mutations elsewhere in our products so it is worth sequencing your whole gene not just the mutation.

Thank you Mrityunjay and Nick. 

Nick are you saying to column purify after DpnI digestion ? I did not perform this. Kindly let me know  that heat inactivation is also required after DpnI digestion ? I performed DpnI digestion followed by transformation. 

I used Q5 polymerase

Details of PCR reaction mixture and conditions are as follows-

I have performed mutagenesis but no able to create mutated DNA. I used 10 ng DNA and same primer as mentioned above. 

Primer (sheet attached also contains PCR cycle ) for quick change site directed mutagenesis (SDM) are exact complementary to each other so I set up 3 sets of PCR reaction to perform SDM.

1. only mutated F primer

2.only mutated R primer

3.Both mutated F & R primer  simultaneously

After this I have mixed independent F & R PCR amplified sample and digested these mixed and simultaneous F&R primer amplified so called mutated samples by DpnI digestion (to confirm DpnI digestion control was setup and after transformation I did not  get colonies for DpnI cut control  sample), then 2ul of this DpnI digested sample were transformed in 50ul DH5alfa  competent cells.

After transformation I picked colonies of these so called mutated samples and sent it for sequencing but I am not getting mutant.

After mutagenesis my sample is wild type (no change in bp) I need to change only 1 bp so during primer designing  I substituted only 1 bp so that I can get change in 1 bp after mutagenesis.

Kindly suggest me how to troubleshoot this ?

Hi Monika

Have you run agarose gel after amplification. Do you get any primer dimer. Also one suggestion you can increase your PCR cycle to 22. I usually do 22 cycles  and has not faced any problem, Also you can keep  Dpn I digestion for more time. Are you using kit or assorted Dpn I and polymerase. In case of assorted buffer compatibility can be a problem. so you have to either keep for long time or as   nick suggest you have to do column purification of PCR product. Check on gel after PCR. Hope these suggestions will help you. All the best.

Ok Mrityunjay thank you. could I get your mail ID?

Kindly tell me is heat inactivation is required after DPnI digestion?

Yes I run gel after PCR for tube which contains both F&R primer not for independent F & R primer tube amplified product and for simultaneous FR primer PCR I am getting band for amplified DNA not for primer dimer.

  Yes Q5 and DpnI are not from kit but both are from NEB (purchased separately)

 kindly tell me is Dh5 alfa ok for transformation after mutagenesis because PCR product have single nick which will be repaired by XL blue 1 super competent cells as mentioned in Agilent/Stratagene technology protocol, but I am using DH5alfa.

Hello Mrityunjay, why PCR purification necessary after amplification, during site directed mutagenese ?

Hi Monika

PCR purification is not necessary if your PCR buffer is compatible with your Dpn I buffer.  In case both are different you can do PCR purification or you can keep the reaction for digestion for a long time. This is because your enzyme may not be very active in PCR buffer and that can lead to incomplete digestion of parental plasmid used during PCR. All the best for you SDM. Tell if you get your mutant. 

Hi Monika,

No a cleanup step is not required (sorry I have too many protocols in my head and this was from another mutation method we use!) and heat inactivation of the DpnI is also not required. As Mrityunjay suggests you could try increasing the amount of template slightly and/or decreasing the primer concentration (what are you using now?) to increase the chances of priming on your template.

We use Pfu turbo and DpnI (from different sources) but we basically follow the Agilent protocols (without the kinase/ligase in the second reaction). DH5alpha should be fine as long as they are high efficiency (>10^8). Check also that your template is not isolated from a methylase deficient strain (then DpnI would be unable to digest it).

I used 10 ng cloned DNA and 0.5 um primer  in 25 ul reaction. Template DNA is pbluescript SK+ which contains my interest of insert DNA.

Q5 pol buffer and DpnI buffer both are from NEB so it should be compatible with each other.

I followed following steps.

1.PCR amplification

2.denaturatiin and renaturation of independent F and R peimer amplified sample( as primers are exact cimplementory to prevent primer dimer firmation i performed independent and simultenous PCR reaction)

3. DpnI digestion

4.Transformation

I have sucessfully isolated so called mutant plasmid from transformed colonies but it is very difficult to select colonies during plasmid isolation. As we do not know which colony is mutated and which one is wild type.

How to identify mutant colonies?

Hi Monika,

First, you said both the Q5 pol and DpnI buffers come from the same supplier, but it doesn't mean they are compatible: you need to check and compare the composition of both buffers (buffer type, pH, salt concentrations,...).

Second, there is no easy way to identify mutant colonies. Sequencing is the only way to verify the mutation. If your mutation add or suppress a restriction site, you can digest  your plasmids and check the digestion profile as a first screening step.