Hi Tomas,

the peak at 230 nm is very likely due to the presence of chaotropic salts like guanidinium thiocyanate. Salts like these are ususally needed for silica membrane based clean up kits like the Macherey and Nagel kit.

High concentrations of chaotropic salts can interfere with downstream applications but the total concentration of chaotropic salts is not very high after elution ( even if the curve looks bad) so your downstream applications might still work.

However, to reduce contamination with chaotropic salts you can do an additional washing step to minimize the contamination (recommended by Machary and Nagel).

Regarding your DNA yield:

Your DNA fragment is relativ big in size - for better DNA recovery heat the elution buffer to 70°C and incubate it for 5 min on the column (recommended by Macherey and Nagel).

Elute the column 2-3 times with fresh elution buffer

Use sufficient amounts of elution buffer (small amounts could interfere with total DNA recovery) (20 µl or more)

Do not overload your column - this might also interfere with total DNA recovery

In addition to measure your DNA with a photometer after clean up I would also recommed to to a gel electrophoresis and estimate your DNA concentration by comparing it to the DNA ladder - at low concentrations and with contamination the results of your photometric measurements might be not very reliable.

I hope this helps

Hi Dörthe,

thank you for your quick answer.

I know that the A230 comes from the chaotropic salts. I actually do three washing steps.

In other experiments, when I need to purify more DNA (like 160 or 200 ul of PCR), I have plenty of gel (e.g. 4x 200 ug). Is this OK to load everything on single column or should I split it? Will it cause more contamination by the chaotropic salts (as the Monarch kit from NEB suggests) or reduction of DNA yield due to some interference?

You are right that I should've eluted the plasmid with heated elution buffer. However, the inserts are 90-350 bp, so they should be fine with RT elution buffer, right? Or does it improve elution of large fragments, but does not hurt elution of small fragments (i.e. it's safer to always heat the elution buffer regardless of fragment size)?

The second elution has always lower concentration, right?

With the MN kit we use 30 ul for elution so that should be fine. We use 15 ul for the Monarch kit, because they say to use 6-20 ul.

Thank you

In addition to Dörthe Andrea Kesper excellent answer I would add that after the extra washes you should elute with 2x the amount of elution buffer recommended and that the buffer should be Hot ( over 70c initially) and leave it on the membrane for 2x as long as recommended before collecting the eluate. Also use low percentage agarose gels and take care not to overload the column

Hi Tomas,

You are right for your smaller fragments the RT elution buffer should be ok - however I don't think that it would compromise your smaller fragments if you heat your elution buffer and you could try this as well.

Regarding your question - if you have plenty of gel I would split it and not load on one column. As also Paul Rutland mentioned overloding of the column might is often not a good idea and NEB is recommending using small gel slices.

They also say, that these salt most likely will not interfere with downstream application (it the same with Macherey and Nagel) - however that you might overestimate the DNA concentration - for this reason I always did an agarose gel in addition. We have used especially the Monarch Kit in the lab (but also Macherey and Nagel) and cloning ususally worked well.

Regarding the second elution: concentration are often not really lower and sometime they could even be higher - so just try it.

And remember you don't need a lot of DNA for your cloning - I usually used 20 ng of vector which mean you would need around 0,4 ng of the 90 bp insert and 1,3 ng for 350 bp an equimolar concentration. This means that you will most likely dilute the concentration of chaotrophic salt significantly in the ligation reaction, even if your DNA concentration is not very high after clean up.

Hiii

I would firstly recommend you to increase the elution time to 30-35 mins and try to incubate at 37'C for better concentration and perform it twice. and during gel extraction washing buffer contains ethanol so try to evaporate it by opening the column for atleast 10 mins before the empty spin.

Hope so it will help.

Hi Paul Rutland

thank you for you insight. Just to clarify - if I use 30 ul for elution, you suggest to load 60 ul of the elution buffer on the column at once, right? Then keep it for 2-4 minutes standing - do you keep it at the heat block or @RT?

Should I do second elution? Also with such high volume?

I used 2% agarose because of the small size of my inserts. But it's true that I could use 1% agarose as I have nice single band. The cut off tiny bits should be removed even in 1% agarose, right?

Hi Tomas,

use either 1x60ul elution or 2x30 ul both hot elution .Theoretically 2x30 should be better. you can use a hot block or just a pcr machine on hold at 70c to heat the elution buffer. It is mainly the initial addition of hot buffer that elutes the dna so the column can stand at RT but transfer the hot buffer quickly to minimise cooling) 0.8% or 1% agarose (preferably low gelling agarose is best) but results are better the less agarose that you use

You can also try the "more is more" approach and start by setting up more PCR reactions, use tape on the gel combs to create really big wells, load all of the PCR reactions. Minimizing the size of the agarose gel slice is really important, so is making sure it's completely dissolved in the first buffer.

Remember, each purification step will reduce your yield. And the concentration of the purified PCR band will be reduced when you set up the digests.

As long as the digests are removing just a tiny bit from the ends of the PCR fragment AND you are using heat-inactivated restriction enzymes, then you can skip the second agarose gel and second purification. All you are doing is losing more product. Use the digested fragments directly for the transformation.

Good luck!

Hi Katie A S Burnette I have one question afterall. I do the "more is more" approach. I actually do 8 PCR reactions (160 ul), tape the combs etc. But then I have the problem with huge amount of agarose gel. How do you handle it? How much of gel equivalent do you load on a single column? As we were discussing with Dörthe Andrea Kesper and Paul Rutland above, it's not good to overload the column, but at the same time, if I split it on several columns, then it doesn't really make sense to make more of the PCR, because I will have low concentration anyway, just in larger volume.

Unfortunately, I'm using BamHI, which cannot be heat inactivated, so I must gel-purify :(

I was considering now to digest directly in the PCR, but I'm using Q5, which apparently is not liked by the restriction enzymes, so that really isn't an option.

I minimize the amount of agarose by using thin gel combs and carefully trim excess agarose with a razor blade. It's OK to use more than one column. See the kit for the exact parameters. You can also dissolve in a larger volume of buffer, spin, & load several times. Using a low percentage agarose gel also helps - I've done 0.8%. It's a bit fragile so you have to be gentle transferring it.

You can always increase the concentration by letting the samples evaporate off the excess water.