Is there any reason for i failed getting any bands on electrophoresis after i did plasmid isolation from E. coli DH5a? My plasmid is ~17 kb in size and i already checked the transformant with colony PCR but strangely i did not get any plasmid band after i isolated them using alkali lysis method even i have confirmed the pellet presence.
I already include RNase A in my isolation protocol so i am quite sure that the pellet was not from RNA. I also have tried to upscale the culture volume to 50 ml and did the midiprep version of alkali lysis. I already reduced the elution volume to 30 ul. I even tried to use Presto Mini Plasmid Kit from Geneaid but i got no result. This is the first time i encounter such problem. If anyone could give me some suggestions i will be very glad.
p.s. currently i don't have access to nanodrop because the facility that has it currently on lockdown
Hi Dylan, It's interesting! I may suggest you check the below points:
- Make sure your pellet is dry and free of ethanol traces before adding water to dissolve.
- Try a control plasmid sample on the gel to see whether the problem is due to the loading dye that you use or not.
- Make sure that you are using the correct amount of loading dye.
- Perform a PCR using the collected plasmid (Pellet).
I would try the extraction again, using a control strain carrying another plasmid, so you make sure the protocol is working properly.
I guess that you were in the plasmid amplification.
How long is your colony PCR product?
You can test your primers using the original plasmid not strains. Additionally, please get your colony PCR product sequenced in order to identify whether your primers' nonspecific annealing.
Did you use restriction enzyme on the plasmid before electrophoresis. Supercoiled circular DNAs travel much faster through the gel then linearized ones. Also what dye do you use for DNA visualization?
Saber Delpasand Khabbazi
- Yes, i am sure it was free of ethanol. We even sniffed the tubes to make sure.
- We also isolated similar plasmid (a bit smaller, ~15kb) and the protocol worked. The one that got problem is the ~17kb ones.
- The loading dye we used is always same, 1 ul for 5 ul sample and 2 ul for 10 ul sample
- We haven't tried the PCR because i am not sure how much template should be added to PCR reaction.
Ricardo Delgado Santander Our plasmid isolation using similar plasmid but a bit smaller in size was succeeded but this one makes me confused.
Sang-Min Paik No, we haven't amplified the isolated plasmid yet. The PCR amplification from colony PCR yields ~400 bp, and i am quite sure with the amplicon size. Unfortunately, sequencing takes a relatively long time in our place so i don't think it's a right choice for a simple confirmation.
Mateo Glumac No, we haven't. We planned to do restriction digest after plasmid isolation but with no band observed we can't proceed with the digestion. We used EtBr for gel staining added directly to the gel. I also tried to soak the gel in diluted EtBr after first observation showed no band but unfortunately the result is same.
My suspicion, the ~17 kb plasmid size is the cause of our problem but i read that we shouldn't get any problem with only plasmid that size. This makes me confused because this is our first time handling plasmid this size.
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