Problems with Dpn1 - (Jul/05/2010 )

Hi,

I want to introduce a single mutation in my gene and does that by using primers with the mutation and with PCR amplify the whole plasmid. The PCR product is then digested with Dpn1 and transformed into DH5a cells. Some of the colonies are cultured and minipreped and the plasmid DNA are sequenced. Sequencing shows around 50% wild type and 50% of my mutant. For me that suggest that it must be something wrong with the Dpn1 digestion. I have tried to optimize the Dpn1 (Used more and more enzyme and less and less PCR product) but nothing works. Can it be something else that is the problem?

A detailed description of my procedure can be seen below:

My Protocol

All enzymes used are from Fermentas.

My PCR reaction:

PCR reaction
dH2O 40,5µl
x10 PFU-buffer+MgSO4 5µl
Start template (20ng/µl) 2µl
dNTP (10mM) 1µl
Primer FWD (100µM) 0,5µl
Primer REW (100µM) 0,5µl
Pfu polymerase (2,5U/µl) 0,5µl
50µl

My start template is plasmid achieved from miniprep with fermentas miniprep kit, eluted in 30μl milliQ-H2O. The plasmid is the vector pSE-420 with a gene insert of 1500bp. Primer used introduces a point mutation in my gene. The PCR amplify the whole plasmid.

The PCR sample is vortexed and runs with the following cycle. 95˚C for 1min, then 18 cycles with 30s denaturation 95˚C, 1min annealing 50.2˚C, 13min elongation 70˚C. After the cycles, the PCR ends with 12min at 70˚C and then cooling the samples to 4˚C.

After the PCR I load 5μl PCR product on 1% agarosgel and run at 110V in 1.5h. The gel shows a distinct band at the size of my amplified plasmid, with indicated that the PCR has worked.

Absorbance measurements gave the below concentration, but that include primer and nucleotide concentrations.

Sample 260nm 280 nm Ratio Concentration
PCR product 0,0978 0,0726 1,35 489,0



After the PCR the PCR-product (30μl) are purified with PCR purification kit from Qiagen (I think you have something similar) and eluted in 30μl milliQ-H2O. (I have not done this PCR-purification step every time, but I did it the last time in hope to get the enzyme to work better.)

Concentration measurements gave
Sample 260nm 280 nm Ratio Concentration
PCR product 0,0051 0,0028 1,82 28,5


So the concentration is much lower after PCR-purification but I don’t think it is so bad cause I had a lot of nucleotides and primers in the sample before.

The sample is then demethylated with Dpn1:
PCR product 16.5μl
Fast digest buffer 2 μl
Dpn1 1,5 μl

The samples were mixed and incubated in heating block at 37˚C in 30 min.

The PCR product is then transformed into CaCl2-comptetent DH5α cells.

Six colonies from transformation are picked, minipreped and sent to sequencing.

Sequencing results show that all three samples are wild type, 50%

Earlier I have not bother so much but now I want to start doing saturation mutations and screen whole libraries and then I cannot accept that it is wild type among my clones, so therefore I really want to find the problem. Can I expect complete digestion of template DNA or do I always get some wild type?

The Dpn1 used is new so it should work properly.

I used milli-Q H2O and not nuclease-free water as Fermentas recommend can that be a problem?

Can it be useful for me to increase the incubation time with Dpn1 even longer to make the digestion complete?

Thanks for any help!

/Lina

Relying on enzymes to completely cut is never a good strategy. But here's some things I would do. First, increase the volume of your reaction, and make the volume of the DNA solution you add a smaller proportion of the mix. Ethanol from your miniprep inhibits enzymes. Make doubly sure the dry spin of the Qiagen kit is done, and is at high speed and long. In my experience, longer digestion times will not change much. I was confused by your concentration measurements. How much DNA are you cutting total?

Was your plasmid template also grown in DH5a? Or another strain?

phage434 on Jul 5 2010, 03:49 PM said:

Thanks for your answer, i will try a larger volume. However I've been in contact with Fermentas and they claim that it should be enough for me to add 1ul Dpn1 to my 50ul PCR reaction, but apperently, it does not work. I always do the dry spin of the qiagen kit, 1min, highest speed, but I can try 2 minutes. Usualy I cut after the PCR without the PCR purifiaction kit, but I tried to use the kit in hope for cleaner sample and then a better cut. Do you think it is a good idea to do PCR purification before cutting or can I skip it?

The first concentration measurment is on my PCR product after the PCR, I did that to get an idea of what my concentration is, and I am aware of that the number include primers, nucleotides and template DNA.

The second concentration measurement is on the PCR product after purification with PCR purification kit, and that should only be my amplyfied product and my starting template. I thougth that the concentration decreased very much, but i've assumed that it was a lot of primer and nucleotids in my sample before purification. Do you think the concentration is reasonable?

Total I'm cutting 16.5ul DNA with the appr concentration 25.5ng/ul

/Lina

leelee on Jul 6 2010, 03:24 AM said:

Yes, my plasmid template was also grown in DH5a. Plasmid achieved by miniprep kit from Qiagen.

/Lina

Hi!
I have been having problems with Dpn1 digestion, se the above discussion and now I got even stranger results.

My problem is that I do not get complete digestion with Dpn1. Of four samples sent on sequencing so is approximately 25-50% wt, which means that Dpn1 digestion has failed. So than I thought that it must be something wrong with my Dpn1 (from Fermentas) so I tested Dpn1 from Stratagene. Then a got gel results according to the attached file. It seems like digestion with Dpn1 from fermentas give a visible PCR sample on the gel while the same PCR sample digested with Dpn1 from stratagene is not visible on the gel. Confusing results for me, does anyone know if it should be a difference between Dpn1 from Stratagene and Dpn1 from Fermentas? PCR product that was not ran in the temperature cycler (No PCR, negative control) is not visible on the gel so it must be amplified PCR product I see on the gel. But then it seems that Dpn1 from Stratagene breaks down PCR product which should be impossible or?

Does anyone know how good cleavage I can expect with Dpn1, how many percent in a sample is wt? I work with saturation mutations and screen libraries so I need to have as less wild type as possible.

Does anyone know any better strategy than Dpn1 digestion to get ride of the start template after site directed mutagenesis?

/Lina