| | Re: Very low plasmid yields from mini-prep
Ok here goes. Its long but thats probably better than many short replies.
I have made mistakes at ALL the following points. This method is for E.coli using a Qiagen kit.
1. Check you have the right strain with the right plasmid in. Streak onto agar with the right selective antibiotic. It happens!!
2. Ensure fresh antibiotic stock, add to the overnight 5ml at the usual concentration and also twice the usual concentration. Plasmids that express betalactamase especially can degrade antibiotic, leading to a mixed culture of with ans without plasmid. If the overnight has grown with twice the normal conc. of antibiotic go with that one.
Please grow to 12-15 hours as stated. Less equals less plasmid, more equals dead cells and genomic contamination. This is important
View 5uL thorugh a microscope. Again useful to ensure you aren't prepping a culture of micrococcus, bacillus and (some) e coli.
3. Miniprep ALL 5ml of overnight, not 1.5ml as suggested. Columns can hold several milligrams of DNA so saturate the column. Spin at 6000rpm for 3 minutes.
For low/medium copy number plasmids use 10ml of culture for ONE column.
For 10ml you'll use 1.25ml Buffers 1 and 2 and 2.1ml Buffer N3.
4. Buffer 1 is a Tris based buffer and must have RNAse added. Resuspend the pellet from the overnight fully. Vortexing at this stage will NOT harm the DNA as it well protected by cell wall.
5. Buffer 2 is NaOH and SDS. It's nasty, caution when handling. Add to Buffer 1/pellet suspension and mix by rolling the tube several times..do NOT vortex at any point from now on.
If using Lyseblue, the whole mass will turn blue.
Do NOT wait 5 minutes until adding buffer 3, when the suspension goes clear that's Buffer 2 finished with. Overdigestion with buffer 2 will damage plasmid DNA. Neutralise immediately after lysis.
6. Buffer N3 is acidic and contains a denaturing agent. Add to buffer 1/2/pellet mixture, rolling the tube until white and no longer viscous.
7. Spin all 5 or 10ml on a benchtop centrifuge for ten minutes at up to 6000rpm.
8. Now split the supernatant into 1.5ml aliquots into Eppendorfs. respin at 13000rpm for ten minutes to remove final flocculant of genomic DNA/cell debris.
9. Run each aliquot through ONE miniprep column. 10mls through one column will truly saturate it.
10. Wash, wash, wash, wash with PE solution. You wont loose any DNA from the wash and the more washes the less conamination downstream. 4 lots of 500ml is fine. Each stage 30s at 13000rpm
11. Dry the column by respinning at 13000rpm for 2 minutes in a fresh Eppendorf. It must be dry to ensure all ethanol has gone.
12. Heat up nuclease free water or TE buffer to around 60 degrees and add 30 uL. Leave for one minute and spin at 13000rpm. Put the ultrafiltrate back through the column and respin.
13. Now add another 20 uL to the column and respin. You will have a total of around 40uL of plasmid.
This method always works for me. Now run on a gel and nandrop to confirm concentration. If the Nanodrop curve is smooth at 260nm its reasonably accurate, else go on what your gel tells you.
With PBAD24 vector I get about 200ng/uL which is plenty.