Hello,
I am currently working on making mutations in my plasmid conducting Quikchange. The plasmid size is 3800bp and I am using primers with several mutations. However, both my parental plasmid and the mutated product have two bands (see attached gel)
The gel shows L1) GeneRuler 1kb ladder, L2) my parental plasmid from a mini prep, L3) the plasmid product (mini product) after conducting PCR -> DpnI -> PCR cleanup -> Transformation -> Mini, L4) the plasmid product (mini product) using the mini product from L3 as template for a new round of PCR -> DpnI -> PCR cleanup -> Transformation -> Mini.
It looks like I get less of my desired plasmid for each round (~3800 bp) and more secondary product. The loss of primary DNA is not feasible, as I need to conduct three consecutive rounds of mutation PCR. Can anyone help me explain what the secondary product may be? I have never seen it before.
The lab works with M13 phages containing ~6400 bp ssDNA. Could the upper band of L2 be the plasmid integrated into phage genome (~10,000 bp total) from previous experiments? And could L3 and L4 be the product of more plasmid integrated into the phage genome? Or is this genomic DNA contamination?
I hope that you can help.
Kind regards Emil
Hello Emil Nielsen
When you run an uncut plasmid on agarose gel, you are most likely to see 2-3 bands. Other than your circular single-stranded plasmid (of expected size), you will also see supercoiled, linear, and nicked plasmid versions of your plasmid. Your gel seems fine. My suggestion is to use 0.8% agarose gel, run at 70 volts for better separation, and load at least 0.5-1ug of the plasmid.
Hope this helps!
Best!
If you think you may have M13 contamination, then transform your plasmids into a non-male host strain (F-) which won't be infected by M13 and then make a new DNA prep.
Pushkal Sinduvadi Ramesh Thanks for the reply. We considered the same - that the secondary bands are alternate formats of the supercoiled plasmid. However, we conducted a PCR using the parental mini product from L2 (see attached) and observed double bands again. PCR products cannot result in alternate formats, right?
Michael J. Benedik Thanks! Indeed, it is a good idea to use a (F-) host strain for future cloning to avoid potential M13 contamination. We suspect that our parental DNA may be contaminated with phage genome. Therefore, we are going to gel extract the parental DNA and retransform this to get "clean" parental DNA.
New gel:
L1: Ladder
L2-3: PCR using mutagenesis primers
L4: PCR using non-mutagenesis primers (control)
L5-7: Same as above with annealing temp of 62°C instead of 57°C.
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