Hi Abeeshna

several potential reasons for poor DNA yield and quality in your plasmid extraction:

Cell Lysis, Precipitation Issues, DNA Binding, Washing Steps, Elution, Nanodrop Measurements, Bacterial Strain , Storage Conditions….

Recommendations:

Review each step of your protocol carefully and ensure that you're following it as intended.

Run a control sample alongside your extractions to compare results.If possible, consider trying another plasmid extraction kit or method to see if you get better results.

Hi Abeeshna!

Well, You can check with the mini kit for plasmid extraction, for example; Qiagen mini plasmid isolation kit. After ethanol precipitation, you must completely dry the pellet to remove any ethanol traces. Also, could you preheat the elution buffer before finally dissolving the DNA pellet?

Hmmm, lots of possible issues. Here are some easy first ones to check:

• Are you using a kit that's designed for that size starting culture volume? It's possible to overload the column with too much cell debris.
• Check the expiration date on the kit & make sure none of the components have precipitated or changed color in a way that indicates spoilage.
• When you say you did "25 ml thrice", do you mean you used 75 ml of cells or that you've repeated the protocol 3 times? If you used 75 ml, then the issue might be the cell volume. If you mean, tried 3 times & still doesn't work, then we have more trouble-shooting to consider.
• Can you get plasmid when you use a smaller volume (1-2 ml) in a standard "mini prep" kit? Or an old-fashioned "boiling lysis" plasmid prep?
• Did you maintain antibiotic selection while growing the liquid culture? Bacteria will "drop" plasmids when there is no antibiotics.
• You certain that you grew up the right cells? Rule to live by if you get cells or plasmids from someone else is "never trust, always verify".

Hope that helps narrow down the list of possibles. Let us know what you try next & what you learn.

Good luck!

Elham Yaghoubi : Thank you so much for your suggestions. I have carefully done each step when i did it for the 2nd and 3rd time . I read the troubleshooting first, read everything on website and opinion of people here in research gate. Still not sure where I am going wrong.

Abdul Rouf

Thank you so much for the response

I am going to try QIAGEN.

I was told that the plasmid size is low and don't need to heat the elution buffer. I would like to try it.

I couldn't even see pellets to discard isopropanol and ethanol. I discarded assuming pellets should be there alongside the wall of the tube.

Katie A S Burnette

Thank you so much for your response.

• Are you using a kit that's designed for that size starting culture volume? It's possible to overload the column with too much cell debris.

This is what the kit says: Grow transformed E. coli in LB medium. Use 15–25 mL (high copy number plasmid) or 25–100 mL (low copy number plasmid) of an overnight culture.

• Check the expiration date on the kit & make sure none of the components have precipitated or changed color in a way that indicates spoilage.

Its all new but I will check again and make sure everything is in good condition.

• When you say you did "25 ml thrice", do you mean you used 75 ml of cells or that you've repeated the protocol 3 times? If you used 75 ml, then the issue might be the cell volume. If you mean, tried 3 times & still doesn't work, then we have more trouble-shooting to consider.

I have used 25ml of culture each time i did the extraction.

• Can you get plasmid when you use a smaller volume (1-2 ml) in a standard "mini prep" kit? Or an old-fashioned "boiling lysis" plasmid prep?

I haven't tried miniprep. I will try again.

• Did you maintain antibiotic selection while growing the liquid culture? Bacteria will "drop" plasmids when there is no antibiotics.

I cultured E.coli with the gene and ampR in 25ml LB broth directly from the glycerol stock and control with just ampR in the same way. I have added ampicillin to the media as well.

• You certain that you grew up the right cells? Rule to live by if you get cells or plasmids from someone else is "never trust, always verify"

I was given the transformed E.coli in glycerol stock. I am volunteering to finish the project along with gaining experience. So I don't have much decision making role.

I am trying QIAGEN tomorrow.

OK!

I'd start with just streaking out some of the E. coli on plain LB (no antibiotic) and on LB + ampicillin to see how it grows. That will let you see if the bacteria have any ampicillin resistance (from any plasmid).

Try and streak to get individual colonies. Ampicillin is notorious for making "satellite" colonies that don't contain your plasmid.

Good luck!

Are the cells a typical strain used in your lab for plasmid prep? If not, it sounds like you got at least a little bit of the plasmid back out. You could transform your lab's usual strain with the bit of plasmid you got then move forward with the new transformants.

Can any of you help me please?

I tried miniprep and got good concentrations.

But my control plasmid is mostly giving 50-70 ng/ul

I streaked colonies from stock. From the agar, I made liquid culture and used 5ml for miniprep QIAGEN.

I also tried growing culture for 16h,17h, and 18h to see the variation.