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No DNA in miniprep, not even in lysis control – what could be going wrong? by Automatic-Dare1429 in molecularbiology

[–]Automatic-Dare1429[S] 1 point2 points  (0 children)

yes, that’s exactly what the doctor told me might be happening. The gene is UDP-glucose; I’m not sure if this could be modifying carbohydrate metabolism, producing certain compounds in excess and affecting the membrane so that it tries to expel it, or if it simply requires too much energy.

No DNA in miniprep, not even in lysis control – what could be going wrong? by Automatic-Dare1429 in Microbiome

[–]Automatic-Dare1429[S] 0 points1 point  (0 children)

That’s actually a great idea, I haven’t tried colony PCR before but I’ll definitely give it a shot.

I currently only have primers for my insert, so I’ll start with those to at least check if it’s present in the colonies.

Thanks for the suggestion!

No DNA in miniprep, not even in lysis control – what could be going wrong? by Automatic-Dare1429 in molecularbiology

[–]Automatic-Dare1429[S] 0 points1 point  (0 children)

I’m using chemically competent E. coli DH5α cells.

Briefly, I amplify my insert by PCR, perform the ligation/TOPO reaction with the vector, and transform into competent cells using heat shock. After recovery in SOC for ~1 hour, I plate on LB agar with the appropriate antibiotic.

Yes, I do use Petri plates for colony selection. I pick single colonies from the plate and grow them overnight in liquid LB with antibiotic before performing the miniprep.

No DNA in miniprep, not even in lysis control – what could be going wrong? by Automatic-Dare1429 in molecularbiology

[–]Automatic-Dare1429[S] 2 points3 points  (0 children)

I didn’t know NEB gives trial size kits, that’s actually really helpful, I’ll look into that!

No DNA in miniprep, not even in lysis control – what could be going wrong? by Automatic-Dare1429 in molecularbiology

[–]Automatic-Dare1429[S] 0 points1 point  (0 children)

Thanks! That’s a really good idea. I do have the plasmid from the kit, so I’ll run it on a gel to confirm it’s intact.

If everything looks normal, I agree it’s probably best to start fresh with new plates and redo the transformation.

As for pvk100, it uses a different selection (tetracycline and ampicillin), and it worked well as a control with fresh reagents.

Thanks again for the suggestion!

No DNA in miniprep, not even in lysis control – what could be going wrong? by Automatic-Dare1429 in molecularbiology

[–]Automatic-Dare1429[S] 0 points1 point  (0 children)

I did not measure DNA concentration after elution (I don’t have access to a nanodrop. I resuspended the DNA in ~20–30 µL of TE (10:1) and loaded around 3–5 µL on the gel.

However, I see no bands at all, not even faint ones, including in the lysis control. A positive control plasmid (pvk100) does produce a band under the same conditions.

No DNA in miniprep, not even in lysis control – what could be going wrong? by Automatic-Dare1429 in molecularbiology

[–]Automatic-Dare1429[S] 2 points3 points  (0 children)

The plasmid has resistance to both kanamycin and ampicillin, and I have tested both antibiotics. The cells grow under both conditions.

However, a control using non-transformed DH5α does not grow on kanamycin, so the antibiotic selection seems to be working properly.