Interesting.

I had never thought about this before. Just following protocols.

It seems normal, now I am thinking.

You have X value of DNA then you digest it therefore you will have i.e X/n concentration base on your product length.

So you have to pickup very higher con of DNA for digestion.

I think but I am not sure as I mentioned.

Regards.

Dear Beatriz,

how long is your PCR product? Do you purify it after the PCR or only after the digest? I recommend to purify it after the PCR to get rid of the polymerase etc. and then check it on gel. If there is still plenty, do your restriction digest (2 hours should be more than enough, usually) and check whether you can heat inactive the enzymes, to prevent the need for a second purification.

Similarly I would keep a part of your purified PCR sample aside and incubate it at the same temperature as your restriction digest (without adding anything else) and load it together with the digested sample on gel to check whether you lost DNA for instance by a DNAse contamination. This should help you to get more insight what your problem might be.

Best,

Jan

Dear Jan-Hendrik Heilers,

Thank you for your quick answer.

My PCR product is roughly 1700pb and after the digestion I would like to purify a 1600pb band, since I plan on losing 100 pb at the end.

I used to purify twice, both after PCR and after digestion, but since I was losing so much DNA I decided to try purifying only after digestion.

Unfortunately neither of my enzymes can be inactivated by temperature since they are BamHI and BglII.

I will try and check on DNAse contamination and I will also try 2h instead of overnight, just in case it´s degrading my DNA?

Please tell me what you think,

Best regards

Beatriz

Generally, I recommend you to purify also after the PCR as the enzymes might be differently affected by the PCR buffer, also you want to have the DNA free from polymerase. If you load PCR before and after purification on the gel, you will already see whether you lose a lot in that step. If this is not the case, you might check the restriction digest with the additional control of no additional enzyme present (but all the other buffers). Then, do your second purification and load a sample of before and after the purification, including the a sample of the initially purified PCR product to conclusively see whether your digest worked. Similarly you will see whether you got a contamination or whether it is the purification that goes wrong.

This approach requires a higher initial PCR product amount, but by this you track every single step and should see when the problem occurs.

Dear Beatriz

which purification kit are you using?

Generally results as your happen for large DNA fragments (>5Kb) while with shorter fragment (generally i'm using the qiagen or the promega PCR purification kits) the yields are around 50-60%

However as i'm doing everytime that here i face some joung scientist facing trouble with restriction enzime, i suggest to you to move to an enzime free cloning approach as PIPE clonnig, which do not require PCR, purification and digestion. It seems a dream, but it is a realitty.

The only limitation is that you need an high fidelity polimerase and original e.coli mach1 cells from thermo (which actually is the only commercially available strain that i know working well with this cloning approach)

You can find more information about it on my blog: ProteoCool ( http://proteocool.blogspot.com/ )

ProteoCool n°1 (Cloning methods overwiev) and

ProteoCool n°3 (Primer Design: PIPE Cloning)

good luck

Manuele

Hello there

In case this helps anyone. I managed to get one successful purification that seems to work when performing the insert-insert ligation test and that I will therefore try cloning today. What I changed was:

- 150 ul of PCR into the same column (instead of just 50 ul) in order to improve final concentration

- When purificating using a column kit, 10 min of incubation time in the membrane/ filter of the column

- Perform this step twice: meaning you collect the elution and put it back in the membrane, wait 10 min again, centrifuge again...etc

- 2 hours digestion at 37ºC instead of overnight, in case there was some DNA degradation going on

- Tried the new T4 ligase and buffer that we bought, just in case the other one had just gotten bad after all the thawing and back to the freezer processes.

My last concentration results for a roughly 1400pb insert were:

- 366.189 ng/ul, purification from PCR (1.92 abs 260/280)

- 207.67 ng/ul, purification of digested insert (1.90 abs 260/280)

Fairly nice resutls :)

Cheers and thanks for the answers

Bea

Hi Bea, I saw your steps for improve the ligation, I have one cuestion:

Do you use the ligation reaction at 22ºC or 4ºC, and how many time do you put the reaction into?

We perform the ligation at 16ºC O/N.