I haven't been able to increase concentrations of my plasmids more than 100ng/uL, I have been using the thermo plasmid miniprep kit. I used 5mL and 10mL cultures, changed my elution buffer to water and still get the same results, which further decrease when I purify my bands from further Restriction digestion.. Any suggestions?
Depending upon what volume you elute in, getting a concentration of 100ng/ul is not bad, you may not get much higher.
If you need higher concentration, just ethanol precipitate and resuspend in a smaller volume.
Increasing the culture volume for a single mini prep often does not lead to an increase because you get inefficient lysis and saturation of the column. You are better off doing independent preps and pooling them, if you need more DNA, then I would just do 10 minipreps and pool them all together and precipitate.
or might as well prepare larger culture and use midi or maxiprep. Also, consider extracting the plasmid without the column if you wish. Then, you won't have the limitation of column saturation. If you need the protocol, here I share the protocol for maxiprep that I used myself:
- Pellet was collected by centrifugation at full speed, remove supernatant.
- Resuspend pellet with 10 mL of P1 buffer and vortex.
---P1 was prepared by mixing 50 mM glucose + 10 mM EDTA + 25 mM Tris (pH 8.0), P1 buffer was stored in 37ºC incubator.
- 10 mL of P2 buffer was added to the suspension.
---P2 was prepared by mixing 0.2 N NaOH and 1% of SDS, P2 was stored at RT.
- The suspension was inverted 5 to 6 times until it turns transparent and slightly viscous.
- 10 mL of P3 buffer was then added.
---P3 was prepared by mixing 3 M potassium acetate + 11.5 mL glacial acetic acid and 28.5 mL demineralized water, P3 was stored at RT.
- Tubes were then centrifuged at 13,000 rpm for 10 min.
- Supernatant was carefully transferred into new tube. Centrifuge again when solid material is found in transferred supernatant.
- Supernatant was split into two 50 mL tubes, each at 15 mL.
- Add 3 volume (45 mL) of 100% cold ethanol (pre-chilled at -20ºC), invert a few times.
- Centrifuge plasmid DNA at high speed (4800 rpm) for 30 min, remove supernatant, plasmid DNA should be visible as white pellet.
- Repeat centrifugation if necessary, when there is more liquid containing plasmid DNA.
- Discard supernatant and air dry for 10 min by inverting tube to allow gravity help to remove liquid and remove excess liquid by tapping onto tissue.
- Dissolve plasmid DNA using water, preferably nuclease-free water, do not use TE when downstream experiment involving enzyme like restriction enzyme.
Modify as necessary if you would like to do mini or midiprep. The difference is only at the volume used for P1, P2, and P3 buffer (I believe you can work it out, refer to the booklet from the kit you bought, I referred it from Qiagen's). Those buffers can be prepared using common lab reagents.
With minipreps it can help to let the column stand with your supernatant for 5 mins to allow the zeolite membrane to equilibrate. Also after the second wash wait 10 mins with the cap off to allow for all ethanol to evaporate. Running your final elution through the column 2-3 times with 2 minute incubation periods can also increase yields about 75%.
Don't use water - pH should be slightly basic. The supplied elution buffer, 1x TE or 0.1x TE would work better.
When eluting, add 20ul of the elution buffer, transfer the columns to 70C heating block for 2-5 min, spin. Add another 20ul (without changing the collection tube), keep it at 70C for another 2-5 min, spin down. You can use lower elution volumes and do 3 elution repeats (I usually do no more than 2). This especially helps with larger plasmids.
Or you can evaporate some water from the eluted plasmid - speedvac, heat block, or vaccum chamber may help.
Hey there! So, you're looking to increase the concentration of your plasmid during a miniprep, right? Well, there are a few different ways you can do this. One option is to start with a larger culture of bacteria that have been transformed with your plasmid. This will give you more starting material to work with, and therefore, a higher final concentration of plasmid.
Another way is to optimize the lysis step of your miniprep protocol. For example, you can try using a different lysis buffer or increasing the amount of lysozyme you add to the lysis step. This can help release more plasmid DNA from the bacterial cells.
Another tip is to purify your plasmid DNA as soon as possible after the lysis step, as prolonged exposure to lysis buffer can cause degradation of the DNA.
Additionally, you can try to increase the amount of salt in the buffer used for purification or increase the number of binding cycles during the purification step. This can help increase the amount of plasmid DNA that is retained during the purification process.
Overall, it may take some experimentation to find the best approach for increasing the concentration of your plasmid, but by trying different techniques and tweaking your protocol, you should be able to get the results you're looking for. Good luck!
Just change the lysis process by the buffer or increased the vortexing time more.
I have two suggestions for this.
1. first elute the plasmid in 30 uL instead of 50 uL
2. If 1 would not work, use the DNA concentrator. It would increase DNA concentration.
Do let me know if these do not work. There are other methods too
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