I ran a gel where what is almost certainly my 8,700 bp plasmid appears to be running slightly heavy versus the 1kb DNA ladder bands. I have been advised at these extremes, the ladder isn't highly reliable. Thoughts? Here is an image of the gel. One lane with a single band was treated with SnabI to linearize it. This may have failed as it should not be lining up with supercoiled plasmid. The other two wells have two bands typical of uncut plasmid and come from different plasmid preps, but appear identical.
regards,
Amy J.
Simply put, they are reliable but a single millimeter difference can represent several kbs. Knowing most gels don't migrate perfectly straight, it is very likely that your ladder migrated a bit further than the more distant wells. To me, your plasmid looks fine. I tend to either use one ladder on each side of my gels, or simply put it in the middle well.
You were right to linearize your plasmid, since the supercoiled form can give you a wrong estimation of the total size. At this size though, you will barely see any difference between the supercoiled and linear forms (see an example in the attached file). Your digestion seem to have worked (providing you do not see any open circular forms if you overexpose your gel).
For high molecular weight plasmids, I recommend you run lower percentage gels (from 0.5% to 0.8%) as it will help separate bands within the 5k-10k range.
Dear Amy
you would probably be advised to run 0.7% gel but regardless at that molecular weight your ladder will only be accurate to about +/- 100bp
For exact sizing I would run a 5% acrylamide gel is exact sizing is required
It is the nature of the colloid that is agarose that means pore sizes are uneven so exact sizing is never possible
I am testing this protocol to use pcr to retarget the above plasmid sgrna, but I get DNA stuck in the well, not migrating. Suspecting very long, heavy concatenation product? I might try 21 cycles and not 30 as they call for.
protocol:
2. Order two 60-mer oligonucleotides, one with the sgRNA guide coding sequence (Step 1with the reverse complement of the coding sequence. 3. Dilute each of the two 60-mers to 100 μM in water. 4. Assemble the reaction in a polymerase chain reaction (PCR) tube: Phusion HF buffer (5×) 5 μL dNTP (10 mM total; 2.5 mM each) 0.5 μL Coding sgRNA guide sequence (60-mer; 100 μM) 0.1 μL Reverse complement of sgRNA (60-mer; 100 μM) 0.1 μL pCAS plasmid 40 ng Phusion DNA Polymerase 1 μL H2O to 25 μL
Perform thermocycling with the following profile, (over 300 minute pcr!) 1 cycle 98 ̊C 1 min 30 cycles 98 ̊C 30 sec 58 ̊C 1 min 72 ̊C 10 min 1 cycle 72 ̊C 10 min 4 ̊C Hold 6. Add 1 μL of DpnI, 3 μL of the provided 10× digestion buffer, and 1 μL of water to the reaction and incubate for 6 h at 37 ̊C (or overnight). 7. Transform the DpnI-treated reaction into E. coli competent cells.
Hi, Amy Johnson sorry for the late response. I was also going to suggest/recommend that you run lower percentage gels, one being the 0.8% which always work best for me. Martin Chenal thanks for sharing. Very insightful!!!
Martin Chenal you might find this interesting https://www.researchgate.net/post/opinions_on_using_PCR_to_re-target_plasmid_sgrna_with_a_universal_sgrna_scaffold
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