Hi,
I have been working on plasmid minipreps using Qiagen miniprep kit and I have run into weird problems of low plasmid yields. When we purchased a new kit, then my plasmid concentrations were 300-400 ng/uL but after that me and I my lab members have used the same kit and we have been getting yields as low as 10-50 ng/uL.
I submit around 250-300 ng of 8 kb plasmid for Sanger sequencing and have been getting no visible reaction or reads only till 150-200 bp. I follow the exact instructions on the Qiagen plasmid miniprep kit. Please guide me in troubleshooting this problem.
Thanks,
Prakriti
Dear Prakriti
first of all, i suggest to you to try the Omega biotek mini kit II, which in my experience is one of the best performing mini kit available.
you can find some data about it in my blog; https://proteocool.blogspot.com/?m=1
ProteoCool n°12
(E.Z.N.A HP MINI KIT II: MIDI KIT YIELDS from a MINI KIT format at page 1
regarding your kit: did you observe visual differences in some specific step? as poor bacterial lysis, different colour of the precipitate after centrifugation.
i can imagine 2 possible problems:
- some buffer contamination o incorrect storage. But to undertand it you have to replace it with new buffer.
- degradation of your antibiotic stock that do not allow propagation od the plasmid during bacterial growth and also if the kit work well, your plasmid yield decrease.
also in this case you have to replace the solution and try again.
good luck
Manuele
Most of the kits are based on the membrane/spin column in which the membrane decide the efficiency of binding and finally the yield. If somehow membrane is not able to bind the DNA, may be membrane is old, dry, ruptured or the whole lot is bad, you can expect you kind of situation. Also, it can happen with kit from any company. In such case, either you can go for kit from another company or do plasmid prep in old school way.
Hi Prakriti Kalra
If you follow the exact protocol of the manufactures' then i would like to suggests you to try another lot of kit or from new company.
Thanks
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