Problems during plasmid prep in cloning - (Jun/25/2008 )

Hi ! I am new here and like the forum and its discussions a lot! I now have a problem of my own. I am cloning a 600bp gene into a pcDNA3.1 vector.I have done something similar to this before and it worked perfectly. But now , I am stuck at the very first step of plasmid isolation! I am using the GE illustra plasmid miniprep kit ( I used the sample kit before and it worked brilliantly). But now after flocculent ppt, when I transfer the supernatant onto the spin columns, I see a lot of bubbles ( which I never used to see before) and after ethanol wash and elution (30ul), the nanodrop shows me values like 6ng/ul,7ng/ul,etc.
Moreover sometimes, when I do get higher yields(according to the nano, which works fine),like around 70-90ng/ul, and run it on a gel, I do not see any DNA!!
I thought this may be due to the LB Amp broth being a little old,(1 month) , so I made fresh stock (Ampicillin-75ug/ml), and yet I have the same problem!! PLEASE HELP!! WHAT COULD BE GOING WRONG???!
Thanks in advance to any and all advice!

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please...any help appreciated!:)

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Hello!!
Have you ever worked with this column?.. Or if the first of the kit?.. I dont think in problems with LB. iT WORKS always.
Have you checked the protocol?.. Did you use this column with a known recombinant plasmid?. Are you sure that you have plasmid??
Did you try another method like alcaline lysis or boliling?

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Yes, I have used this kit before and it has worked beautifully...I follow the protocol exactly as described..it has never troubled me before...I am trying out the alkaline prep with home-made buffers now..thanks for the reply!

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I haven't used the GE illustra kit before so I don't have any experience with it. However, I would check the expiration date of the kit as I've come across buffers that go bad after a while. Exposure to light and heat isn't a good thing either. Anyways, what you can do is try a different kit. I frequently use the Zymo Research Zyppy Plasmid Miniprep Kit. Right now, you can order a 50 prep kit for free! (I ordered a few myself).

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Hey, thanks for the suggestion! We actually have a couple of free ZYPPY kits but was not sure how well they work..Since you have used it, how good is it? Good yields??

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The yields are very good. Depending on the copy number, I've consistently recovered 1.5-5ug of plasmid DNA. I've made a few modifications to the protocol myself though. I do not recommend the direct LB extraction method though. The LB seems to decrease yields significantly. So, what I do is take 3mL of overnight culture, and centrifuge the cells down. You can use a lower volume if you wish but it's important to remove the LB. I use a 1.5mL microcentrifuge tube so it take two 30sec spins at top speed. Discard the LB and resuspend in 600uL water. Proceed with the lysing according to the kit and centrifuge the cell debris for 3 mins (3 mins is sufficient to allow the RNase to do it's thing otherwise you see a faint smear on the gel from the RNA). After loading the supernatent onto the column, I spin at 8,000rpm for 2-5 mins to bind the DNA instead of the >10,000 rpm the manual says. At higher speeds, the DNA will elute through a bit. Just make sure all the liquid flows through the column. Wash using buffers and then I use 50uL of sterile MilliQ water to elute the water. Make sure you let the water soak onto the column for 1 min before doing the final spin. You can also to 2 x 25uL of water to slightly improve DNA yields. I don't like using the elution buffer since it can it can interfere with down stream processing like restriction enzyme digest but more importantly it can greatly affect PCR.

If you are planning on sending your sample for sequencing, I recommend using the Zymo DNA concentrator column to further purify the DNA and concentrate the DNA further.

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Hey! Thanks! I used the zyppy kit and it gave great yields!! ~360 ng/ul Can i use the zyppy dna concentrator for dna purification from gel slices? the protocol mostly talks about pcr preps...

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