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Appearance of a higher molecular weight band after plasmid isolation? (NOT chros

Plasmid isolation Lentiviral vector Miniprep

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#1 DaRQsiDe

DaRQsiDe

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Posted 02 January 2014 - 07:34 AM

Hello everyone,

 

I have a problem that I have never encountered before... I am cloning a couple of shRNA designed by the TRC guidelines to pLKO.1 vector. I constantly encountered this problem - I have a higher molecular weight band with the plasmid preps that I have done. I have tried Roche High Pure kit, isopropanol precipitation, phenol/chloroform extraction and PEG-precipitation as techniques and DH5a and Stbl3 as different strains. Previous colony PCR gave positive results but I could not get plasmid. Recently, I used the Macherey Nagel Nucleospin kit and obtained high-molecular weight band free plasmid...

 

Gel 1.png

 

1 st lane is Fermentas GeneRuler Mix ladder, others are the shRNA directly after mini. The band is higher than expected but I have observed the same band in the Thermo Scientific "The RNAi Consortium (TRC) Lentiviral shRNA Technical Manual" (Figure 4), so it was allright. I put those into fridge.

 

Two days after (New Years' Eve and such), I tried to do a restriction digest with those. The gel is below. Upper row in the first gel is the left of the second ladder in the second gel, and lower row in the first gel corresponds to the right of the second ladder in the second gel.

 

Gel 2.png

 

Here, the plasmid band disappeared, and a higher molecular weight band appeared, which cannot be cleaved by the restriction enzyme pair that I previously used for screening pLKO.1 inserts (SpeI/NdeI). As controls, I had the "empty" vector that I have used for cloning (without the stuffer but still produces compatible ends), which is purified using Roche Genopure Midi Kit previously and there were no problems...

 

I am really perplexed by this, in my six years doing PhD I did not encountered any problems in cloning and now in my first post-doc I feel like I hit a brick wall. If you can provide me with any explanation on what may be going wrong I will be really glad.

 

Thanks,

 

Cihan Aydin







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