I'm currently trying to assemble a plasmid with my insert. The plasmid is the SAGFF (6293bp) and my inserts I want to insert (individually) are 2300bp and 3000bp.
I have used both a 1:2 and 1:3 ratio, but my minipreps are negative for inserts when digested with enzymes.
I have a feeling that it's a concentration problem which is leading me to assemble with faulty concentrations. I suspect it's our photometer and unfortunately, the nanodrop is also out of order at the moment. The readings I'm getting are on average at a 1:200 dilution
0.8 ug/ml for the 6kb plasmid
0.3 ug/ml for the 3kb insert
1.1 ug/ml for the 2.3kb insert
I'm assuming these values are quite high after PCR purification and the photometer is giving inaccurate readings. The concentrations agree with my gel electrophoresis as both the plasmid and 2.3kb insert showed strong amplfication.
Any suggestions? Should I try 1:3 ratio and/or run it longer?
Thanks!
Hey Andrew,
Your concentrations may be the issue. I have found I need really sharp and strong bands for GA to work. Your concentrations actually seem quite low to me. I would say that you should be hoping to get over 30 ng/ul for your PCR products. I have some questions;
1) Do you have strong single bands (with no non-specific bands) on your gel run out?
2) What are your 260:280 and 260:230 ratios?
3) How long are your overlaps?
4) Have you purified your PCR products with a good kit (eg Qiaquick)?
5) Are you using miniprepped vector backbone? perhaps midiprep it (to reduce endotoxin)
It seems like you'll need to wait for your nanodrop to back up online (or is there a bio-analyser at your institute?) to get accurate concentrations and ratios. But running on a gel should tell you the concentration and whether there are any non-specific bands.
I would also suggest you run your assembly on a gel after you've incubated at 50˚C for 60 minutes so as you can get an idea if there has been any assembly. If it is a case of there being only a small amount of assembled product, which is not enough to transform properly, then it may worth your while amplifying the assembled product using complementary primers that cross the border of your two insert boundaries, then transforming that.
In my experience with GA, your DNA has to be perfect for it to work! Hope my tips help!
I would recommend estimating DNA concentration by comparing band intensities (band of interest vs DNA ladder - DNA amount is typically given for each band in DNA ladder) after electrophoresis and EtBr (or similar fluorescent dye) staining. Even it seems inaccurate, it is superior to UV-spectrophotometry (Nanodrop etc), which gives false high conc if proteins, primers, dNTPs, nonspecific products etc... are present present in your sample.
Your concentrations (for example, 0.8 ug/ml = 0.8ng/uL) looks very low . Did you observe your target DNA on agarose gel? how much did you load on the gel? I am afraid that you might have detected the band that corresponds to primers or primer dimers. I personally recommend to repeat your experiments with appropriate controls. Run primers on the gel as controls.
Thanks for the replies and suggestions. To answer a few:
Overlaps are 18bp long
Fragments and plasmid were purified with Qiaquick
https://imgur.com/a/6ECDw
That is the gel I ran with ladder - plasmid - plasmid dilution - plasmid digestion - three inserts
They are the correct size, so I believe amplification was succesful
I used another spectophotometer and got higher readings (some not making sense)
at 1:100 dilution, I got readings (7.1 , 9.1 , 19.5 , 30.6)
The 30.6 is not the last band that has high amplification, that one was 9.1 ug/ml.
I am running a new gel to try the DNA ladder comparison. How much of the sample should I load into the wells? Also, I believe I need to dilute one of my inserts, so what dilution should I do (1:10)?
You can try loading more dilutions - bands in the ladder and bands of interest should be approximately of the same intensity. Do not overload it - the range between 10-100ng/band is typically optimal.
Eg: Diluted plasmid and first (or first two) insert seem to be ok, you need to dilute ladder and the third insert, (and possibly increase the amound of the second insert) .
Alright thanks everyone. Seems the problem was DNA purity as I reamplified a new set and got about 40-100ng/ul concentrations with 1.8-2.0 absorbance ratio. We ran out of Gibson master mix though, but used TOPO blunt and it worked.
Gibson assembly requires really pure inserts... lesson learned.
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