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Low DNA Extraction - (Feb/15/2007 )

hi i've been having some problems with obtaining high yields of DNA from my DNA extractions using QIAprep Miniprep Kit
I'm extracting DNA from TOP 10 E.coli and i am getting ~12.5 - 20 ng/ul of DNA from my extracts
When I run pcr on my extracted DNA i get multiple bands in my gel (my target fragment that should be around 1500bp) none of which are at 1500bp
Right now the only thing that i could think of that is causing this problem would be that I am over incubating the E.coli when i do a cell culture ~20 hours instead of the prescribed 12 -16 hours. but does the incubation time make that much of a difference?

Any suggestions? thanks

-cheesecake-

Hi, just a couple of suggestions for you -
You may have some antibiotic-resistant bacteria contamination, which is why you get cell growth but not the plasmid you need.
You are using a low-copy number plasmid, which means you're not purifying much DNA, and your PCR isn't working very well.
The kit is old and some of the reagents may have gone off?
You're being too rough during the cell lysis step, and are co-purifying bacterial genomic DNA, which is interfering with your PCR.
How much culture are you using for the purification? What plasmid and antibiotic are you using?
A restriction digest would probably be better than PCR to identify your plasmid, perhaps try that.

-scrat-

QUOTE (cheesecake @ Feb 16 2007, 12:18 AM)
hi i've been having some problems with obtaining high yields of DNA from my DNA extractions using QIAprep Miniprep Kit
I'm extracting DNA from TOP 10 E.coli and i am getting ~12.5 - 20 ng/ul of DNA from my extracts
When I run pcr on my extracted DNA i get multiple bands in my gel (my target fragment that should be around 1500bp) none of which are at 1500bp
Right now the only thing that i could think of that is causing this problem would be that I am over incubating the E.coli when i do a cell culture ~20 hours instead of the prescribed 12 -16 hours. but does the incubation time make that much of a difference?

Any suggestions? thanks



Incubation time makes a difference. For some plasmids types and unstable inserts it makes the difference between sucess and failure. The same can be said with incubation temperature. At high temperature (37 Celsius), some unstable inserts will start rearranging. The cure is to grow the cells at 30 Celsius.

Multiple bands in PCR is not diagnostic of failure. However failing to get the expected bands is not good. I would also say, you should check you DNA by restriction digest rather then PCR(which might has a chance of failure, or experience problems)

Now if your kit is good, I would suggest you try growing your cells at 30 Celsius. And perhaps grow more a large culture volume. Grow 2 flaskes of cells. And keep to 14-16hrs of incubation

-perneseblue-

QUOTE (scrat @ Feb 15 2007, 02:27 PM)
Hi, just a couple of suggestions for you -
You may have some antibiotic-resistant bacteria contamination, which is why you get cell growth but not the plasmid you need.
You are using a low-copy number plasmid, which means you're not purifying much DNA, and your PCR isn't working very well.
The kit is old and some of the reagents may have gone off?
You're being too rough during the cell lysis step, and are co-purifying bacterial genomic DNA, which is interfering with your PCR.
How much culture are you using for the purification? What plasmid and antibiotic are you using?
A restriction digest would probably be better than PCR to identify your plasmid, perhaps try that.


i don't believe i have antibiotic-resistant bacteria. my plates are ampicillan plates that are pretty fresh and the way the bacteria colonies formed on my plate are consistant with my streaking technique
The kit is relatively new (within 6 months)
hope i am not too rough on the cell lysis, will be mindful of this
I'm doing a 5 ml culture using TOPO Vector and ampicillian as my antibiotic
can't run a digest if my DNA extraction is too low. nothing will show up when i run the gel

thanks

-cheesecake-

Miniprep manual did mentioned something like this. Can't grow more than 17 hours or else the plasmid will somehow degraded together with the cells.

I do agree with perneseblue, grow more and spin down in the same tube. Lyse together and hopefully you will get more plasmid out from there. wink.gif

-timjim-

Use 15uL of your prep for a restriction digest, that will be between 180 and 300 ng per digest, which will be quite easy to see.

-scrat-

once i minipreped 24 hours cultured bacteria. the extracted DNA was OK.

-ligation doesn't works-

Hi,
It has happen to me before and I don't know what was the problem. I was using TOPO TA vector to ligate with the PCR product. When I transformed DH5 alpha the cells grew well on a plate but in liquid medium (LB+ Amp) they didn't grow that much, so I didnĀ“t get much DNA extracted with the QIAGEN kit.
Since TOPO vector carries Kannamycin resistance marker as well as Ampicilline I grew the colonies in liqid medium plus ampicilinie and kannamycin Grew more cells and lyse them. At the end, elute DNA from the column in a small volume of water.

-RMG-

What do you pick your cultures with? (pipet tips are best imho)
What media are you growing your cultures in?
Our lab uses TB with 1g/L dextrose. I can get up to 2ug of DNA from a 1.5mL culture using a homemade protocol with Qiagen buffers. Very simple.

Pellet bacteria in 1.5mL eppendorf, asp SN. Resusp in 300uL P1. Lyse w/ 300uL P2, neutralize w/ 300uL P3, inc on ice for 10". Spin @ max speed for 8'. Xfer SN to fresh tube, respin. Xfer SN to a fresh tube containing 630uL Isopropanol. Spin 8" @max speed. Wash w/70% EtOH. And if you resuspend in milliQ H2O, it's ready for sequencing.

Hope something here helps. We had the same issue until I made hte lab swtich to the above. Worked like a charm for us, hope it does for you.

-plays_with_ecoli-