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Qiagen HiSpeed maxiprep - precipitants and transfection troubles

Maxiprep DNA isolation Transfection

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#1 DrSnood

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Posted 16 February 2017 - 01:18 PM

Hi all,

 

Wondering if anyone has experienced issues with downstream applications using DNA isolated with Qiagen's HiSpeed maxiprep kit. I'm using this kit to prep plasmids (backbone = pcDNA3) and then transfecting them into HEK293T cells for luciferase assays. Here is where strange stuff is occurring.

 

Backstory: Our lab switched to the HiSpeed kit about 4 months ago. Prior to that, we were using Qiagen Maxiprep kits. I dove into a bunch of experiments to determine the effect of my protein of interest (POI) on transcriptional activity of transcription factor X. Everything was working really well. I then cloned a bunch of truncation mutants to determine the domain of my POI involved in this process and prepped them with the HiSpeed kit. Again, everything working great. One month in, everything crashed - wacky Renilla values (low, erratic), poor expression. After doing a lot of troubleshooting, I re-prepped all of my plasmids out of total desperation. Issues resolved, everything was working. One month later (this week), I generated a new point mutant and put it into my luciferase system along with the plasmids I prepared a month ago. The issue is back - my Renilla values from cells expressing any of the old plasmids (there are 3 - negative control, full-length protein, truncation mutant) were ~10-fold lower than those obtained from my cells expressing the point mutant with the newly prepped plasmid. I did a Western to check protein expression, and saw a similar pattern with my POI, with the old plasmids giving ~10-fold less expression.

 

Some observations: The Qiagen HiSpeed kit relies on a precipitator disk rather than isopropanol to isolate the DNA. The disk is then washed with 70% ethanol a few times and the DNA is elute with TE. We have noticed while precipitants in these preps. I reprecipitated my DNA with sodium acetate and washed it a few more times with ethanol, but the precipitants remain. We have also tried eluting from the HiSpeed column after the washes with QC and doing an isopropanol precipitation instead of the disk - same outcome.

 

My question: Has anyone seen these precipitants in their preps? Have you had issues with plasmid stability with constructs isolated with this kit? Any ideas on WTH is going on, and how I can address it? (Yes, the most logical answer would be to go back to the regular Qiagen kits, but I have to prove to my boss and lab manager that the kit is the problem because she bought EIGHT boxes.)

 

Thanks, everyone!







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