Looking at other questions and answers regarding typical cloning procedures (restriction-ligation), I notice almost everyone suggests to GEL purify DNA at several steps. For example ; after a PCR amplification and prior to restriction digestion ; or after a restriction digestion and prior to ligation.
Knowing gel purification often leads to the loss of >90% of initial DNA, as well as making it near impossible to evaluate quality and quantity of DNA by nanodrop measurement, why would it be recommended over a simple PCR-purification (column) ?
The way I see things, PCR-purification will remove primers (from PCR), short DNA stretches (from digestion of a PCR fragment), and will also change the buffer to a neutral one. Also, one can always run a few microliters of a PCR product or vector linearization on a gel to check for purity/complete digestion, and then do a PCR purification on the remaining volume.
Note : I am aware gel purification is essential when removing a chunk of a plasmid by digestion. However, I don't see the point when the vector is only linearized or the insert come from a PCR amplification.
What is your input on this topic ? Gel-purif or PCR-purif ?
Hello Martin,
I tend to stick to gel purification for PCR fragments to be inserted into a vector by restriction for two reasons:
a) If the amplification wasn't fully specific and if fragments smaller than the desired products are generated, these will get cloned preferentially, leading to a large proportion of spurious clones.
b) With a preparative PCR (vol 100 µl), it is quite common to recover 1-2 µg in a volume of 30 µl after gel purification, which is plenty for performing the restriction digest, and quite quantifiable on a Nanodrop spectrophotometer if you take care of eluting the DNA from the minicolumn in a small volume (e.g. 20 + 10 µl). In fact, you should not forget that, since PCR fragments are usually short (0.5-3 kb), even a low concentration in terms of ng/µl represents a relatively high molar concentration. Thus, by adding too much material , you risk incomplete digestion by the restriction enzymes.
However, after performing the digestion, it is quite OK to use the PCR purification protocol.
As to running an analytical gel to check the quality of the PCR amplification, if taking the trouble of running a gel at all, I prefer to run a preparative gel that will both tell me if the fragment is clean and give me the opportunity to purify it if it is not.
Hello Martin,
I tend to stick to gel purification for PCR fragments to be inserted into a vector by restriction for two reasons:
a) If the amplification wasn't fully specific and if fragments smaller than the desired products are generated, these will get cloned preferentially, leading to a large proportion of spurious clones.
b) With a preparative PCR (vol 100 µl), it is quite common to recover 1-2 µg in a volume of 30 µl after gel purification, which is plenty for performing the restriction digest, and quite quantifiable on a Nanodrop spectrophotometer if you take care of eluting the DNA from the minicolumn in a small volume (e.g. 20 + 10 µl). In fact, you should not forget that, since PCR fragments are usually short (0.5-3 kb), even a low concentration in terms of ng/µl represents a relatively high molar concentration. Thus, by adding too much material , you risk incomplete digestion by the restriction enzymes.
However, after performing the digestion, it is quite OK to use the PCR purification protocol.
As to running an analytical gel to check the quality of the PCR amplification, if taking the trouble of running a gel at all, I prefer to run a preparative gel that will both tell me if the fragment is clean and give me the opportunity to purify it if it is not.
If the PCR products appear clean by gel electrophoresis, we can often succeed with PCR purification alone. Make sure you stop the gel early to catch small fragments, such as primer dimers, before they run out and thus become invisible. Confirmation by sequencing should be mandatory in any case. If it's the right sequence, it won't matter how you got it.
You can always fall back to gel purification if you experimentally determine that you are in fact cloning shortened products. In my experience this is going to be quite rare, but it does happen. If gel purification is difficult for you, I think it's OK to use PCR purification as your "plan A", assuming your band is clean.
But it does not need to be difficult. Often possible not to lose 90% during the gel preps. Make sure the well is large -- fill it no more than half. Otherwise it can easily run out into the buffer, because the gel near the well tends to be curved up, making the rim of the well taller than the rest of the gel is. Load the melted product onto the column twice or three times. And elute twice too, with heat. Starting with a large amount of DNA certainly helps as well.
Hi John,
I have been using Axygen beads for most of my purifications lately. The ratio of beads to starting material gives you the ability to impart size selectivity, It is possible to select both upper and lower size limits and is used extensively to select insert size distribution for NGS library preparation. I use it these beads for most of my post reaction clean up or size selection now across the board and they have worked well for me.
I hope this is helpful.
Frank
I understand your question and tbh I used to feel the same way. I referred to many protocols, and what I found was the ingredients of PCR (especially the salts and the buffer) can really hurt the efficiency of restriction digestion/ligation. And yes, there is a clear loss of sample DNA in these purification steps. But you can always compensate for the loss by taking a higher concentration of starting materials. I have always stuck to Thermo Scientific GeneJET Gel Extraction Kit (Catalog number: K0691). Its been very reliable for me, and as I said if you take the PCR ingredients at higher concentration, the loss ain't that much.
Hi,
If the PCR Product is clean with single band, PCR purification Kit can be used. As you mentioned recovery from gel is very low.
Hi,
In order to minimize the loss of your PCR product you can use PCR purification kit from Qiagen or Thermofischer. This method can be used provided your primers are specific and give single band on the agarose gel.
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