10 replies to this topic

[Ikar]

Hi,
my lab colleagues and me are all using the same Kit (Peqlab) for our minipreps of plasmid DNA from
bacteria cells.
While they are getting yields >100 ng/µl out of their bacteria I only get half of it or even worse.

My protocol is based on the manual which comes with the peqlab miniprep kit:


1) Pick 1 colony from selection plate and dilute it in 10 µl H2O.
2) Use 0.5-2  µl for a colony PCR test.
3) Add the rest  to 5 ml growth media (+ antibiotic) and incubate overnight at 37°C.

4) Pellet Bacteria by centrifuging at 4,000 rpm for 10 min; compl. remove supernatant completely
5) Resuspend pellet in 250 µl Solution I/RNAseA by pipetting
6) Transfer resuspended bacteria to a 1,5 ml reaction tube
7) Add 250 µl Solution II and gently mix by inverting; incubate 1 min at RT
8) Add 350 µl Solution III and gently mix by inverting
9) Load DNA binding column with max. 750 µl lysate; centrifuge 1 min at 10,000 rpm
10) Reload flow-through onto the same column again and repeat centrifugation
11) Remove flow-through and add 750 µl DNA Wash Buffer to the column
12) Centrifuge 1 min at 10,000 rpm
13) Remove flow-through and add 750 µl DNA Wash Buffer to the column
14) Centrifuge 1 min at 10,000 rpm
15) Remove flow-through and centrifuge 1 min at 10,000 rpm to dry the column
16) Put DNA column into a new 1,5 ml reaction tube and elute DNA by adding
        70 µl H2O and centrifuge 1 min at 5,000 rpm

Do you have some tips for me how I can improve the yield besides lowering the volume of water
for elution in step 16?

I'll be very thankful for any kind of tip!

[phage434]

Between steps 8 and 9 are you spinning down the cells and loading only the cleared lysate?  This is important, since otherwise all of the cell debris is loaded on your column. If you spin down after 8, the debris is left behind.
5ml of culture is a lot for good lysis, assuming the cells grow well.  You might be using too many cells for good lysis.  Try 2-3 ml of cells to start with.
You can and should be vigorous in resuspending cells in step 5.
You can elute twice, first with 40 ul of TE, then an additional 30 ul of TE.  Use TE rather than water, which will protect your DNA, and have little effect on downstream digests or sequencing.  Pure water also has uncontrolled pH, and may be acidic enough to not elute DNA effectively.

[Ikar]

Thank you so far

Between step 8 and 9:
Of course I'm spinning down the SDS-debris...it's a typing error. I centrifuge for 10 min at 10,000 rpm (tried both: room temperature and 4°C and observed no differences).
I also tried either to decant the supernatant or to transfer it carefully with a pipette... I think decanting works a little bit better.

To the 5 ml culture:
Yeah, they grow very well. Maybe I should keep growing them in 5 ml and after growth separate them in 3 1.5 ml reaction tubes (500 µl which are left over are used for a glycerol culture).

To the elution:
The idea with TE sounds convincing. What pH should it have? pH 8?
Does the TE have negative effects on PCR reactions or sequencing approaches?

Edited by Ikar, 23 April 2011 - 09:23 AM.

[perneseblue]

EDTA does chelate Mg ions. And this activity could in theory inhibit down stream enzymatic reactions. However in practice, this doesn't really occur, as the amount of DNA use is small and thus only a small amounts of EDTA is present. PCR reaction usually have a surplus of Mg ions. And for sequencing purposes, you often have to dilute your DNA as it is too concentrated. Use water to dilute an aliquot of the DNA sample.

If you are concern, you can use Tris-HCl rather than Tris-EDTA (TE) to elute your DNA.. But EDTA does prevent any contaminating nuclease from degrading the DNA.

[pDNA]

dont worry to much ...if your collegue and you are working on different plasmids.

Different plasmids can really differ in their yields (although they have the same origin of replication). The sequence of your insert can e.g. drastically interfere with plasmid replication or you have promoter activity that leads to head-on collisions during transcription and replication lowering your replication frequency.

So don't worry to much ...make a competition using the same plasmid and strain and the same culture and compare your yields ...if they than differ significantly ...start having sleepless nights and try to erredicate your systemic error

Good luck!

Regards,
p

[Ikar]

About the TE buffer thing: What about storing primers in TE? Would it make sense to dilute them in TE
instead of water?

What TE pH would you recommend for DNA storage?
I found different recipes on the internet:

a)
10 mM Tris-Cl, pH 7.5
1 mM EDTA, pH 8


10 mM Tris-Cl, pH 8
1 mM EDTA, pH 8

[perneseblue]

Master stock (100uM) of the primers are usually made with TE.
An aliquot of the master stock is then diluted with water to make the working primer solution (10uM)

For DNA, TE at pH 8 is used. While for RNA, TE with pH 7.5 is used.
Why? Well it is said that DNA is more soluble in a slightly alkaline solution and RNA more soluble at neutral pH.

I have not seen the difference, though. So is this bit of "lab wisdom" actually true?

And lastly... in addition to phage434 suggestion,  if you heat your TE, (~70 C)  you can elute more DNA from the column.

[phage434]

I always store primers in TE as well.  TE does two things.  First, it keeps pH above neutral, which stabilizes DNA.  pH 7.5 or 8.0 is a tossup here, and is controlled by the tris pH.  The EDTA chelates out magnesium and other heavy metal ions.  Magnesium is a required co-factor for nearly all DNAse enzymes, so that even if your DNA is contaminated from fingertips, e.g., the enzymes will be inactive.

Now, about the use of DNA stored in TE.  The amounts (volume) of DNA solution added to reactions is usually quite small (if it is not, that is often a problem in and of itself, for other reasons).  A typical restriction enzyme buffer at 1x concentration contains 10 mM magnesium.  If you add half of the volume as a TE containing solution, then you have adjusted to 0.5 mM EDTA, an inconsequential amount, leading to a minor change in the net magnesium concentration.  For primers at reasonable concentrations, a similar situation arises.  You usually set up a pcr reaction with 1 ul or less of each primer in a 20-50 ul reaction, and similarly low amounts of template.  These amounts will change the reaction conditions very little.  Sequencing centers often tell you not to submit samples in TE, but in all the years I've been sending them, I've always sent DNA in TE, and they have always worked (well, the ones that should have).

About the pH.  As far as the DNA goes, it is a don't care.  For elution from columns, however, there can be a pH dependence.  Here, look at the column protocol.  Usually pH 8.0 is better.  This is a reason for using TE (or at least Tris buffer), since pure water often has an acidic pH from absorbing CO2 from the air.

Some people use TE 10:0.1, or other mixtures, containing less EDTA.  I used to do this, but no longer see the point.

[Ikar]

perneseblue, on 24 April 2011 - 04:28 AM, said:

Master stock (100uM) of the primers are usually made with TE.
An aliquot of the master stock is then diluted with water to make the working primer solution (10uM)

For DNA, TE at pH 8 is used. While for RNA, TE with pH 7.5 is used.
Why? Well it is said that DNA is more soluble in a slightly alkaline solution and RNA more soluble at neutral pH.

I have not seen the difference, though. So is this bit of "lab wisdom" actually true?

And lastly... in addition to phage434 suggestion,  if you heat your TE, (~70 C)  you can elute more DNA from the column.

In our lab everyone makes the master stock (100 µM) with dH2O - as far as I know. And since I began working there I've been told
to do so, too...
I've to mention that the primer master stocks are stored at -70°C so maybe (in opposite to a -20°C storage) there is no need
for using TE!?

Would you recommend to make my dilution stock (5µM) with TE from the master stock (e.g. 950 µl TE + 50 µl primer) or would the
pretty high TE concentration have negative effects on pcr and sequencing reactions?

EDIT:
I have just been told on the web page of our sequencing service that there is no way in using TE as dilution buffer for DNA probes to be sequenced.
That would restrict my range of choice to Tris-HCl buffer, but better than pure water anyhow...

EDIT2:
If I am planning to use Tris-HCl for an elution and storage buffer what concentration should it have? 10 mM or something like this?

Edited by Ikar, 24 April 2011 - 11:36 AM.

[perneseblue]

I would not recommend using TE to dilute the stock primer to make the working primer solutions.  The TE in such a situation would be of little use in keeping your stock primers safe and opens up a small chance that the EDTA could inhibit down stream PCR reactions.

The purpose of EDTA  in TE is to inhibit nuclease activity when the stock solution has been thawed. So keeping the 100uM primer stock at -80 C doesn't actually invalidate the need of using TE. More over the Tris in TE helps maintain the pH. Water tends to be acidic due to atmospheric carbon dioxide.

10mM Tris-HCl should be okay. That is what most often is used.

[Ikar]

Some feedback on my recent minipreps:
They are becoming better and better (the yield)!
Thank you for your tips.

But now I got another question:
Would a 4°C centrifuge account for a better precipitation after adding the neutralization buffer?