Objective: Stuff 8 sgRNAs (~ 0.4 kb/each) into my vector (~ 12 kb) using Golden Gate cloning
Problem: while the experimental plate shows plenty of colonies, enzyme digestion of the plasmids extracted from the overgrown colonies in LB tubes yields NO band.
Additional observations:
1. Gel electrophoresis of uncut plasmids displays presence of SHARP bands of relaxed and supercoiled plasmid DNA. In usual case, the lower signal is a bolded rectangular shape rather than a sharp line due to high concentration of DNA.
2. Change of a different digestion enzyme pair resulted in the same NO band result.
3. Plating of empty competent cells yielded no colony suggesting that competent cells are not antibiotic resistant.
4. Plating of negative control (contains every component except the sgRNAs) yielded 3 colonies suggesting that the majority of the colonies on the experimental plate is not the uncut vector.
5. Plasmid yield is always at ~ 60-90 ng/ul even when the same colony was overgrown in 3 LB tubes and later pooled together.
6. Centrifugation of the overgrown culture yielded the usual-looking amount of cell pellet.
Experimental details:
1. The primer pairs for sgRNA PCR are designed using an online tool specific for Golden Gate assembly provided on NEB. The tool appoints the overhand sequences, BsaI recognition sites, and additional randomized bases at the ends of the primers to allow sufficient enzyme digestion.
2. Each sgRNA is PCR amplified from the templates using PrimeStar MAX DNA Polymerase Master Mix (Tanaka) in 2 reactions and pooled together into one column to yield concentrated amplicons (~ 150 ng/ul). Shortly, there are 8 amplicons.
3. The 8 amplicons and the vector plasmid were mixed together along with Golden Gate buffer and Golden Gate master mix (NEB) as instructed in the manual follow by thermocycling program for 9 fragments.
4. 2 ul of the GG reaction was used to transform 50 ul of NEB Stable High Efficiency Competent Cells with routine heatshock procedure and a 30-minute recovery. 100 ul of the recovered cells was plated on an Amp plate and cultured at 30oC for 18, 20, 22 hours (yielded no different outcome).
I would like to hear from your point of view what could possibly create this scenario where cells can grow on Amp plate, in Amp LB tube but seem not to contain a decent amount of and accurate plasmid.
Regards,
Albus Truong
It is difficult to diagnose an experiment from afar, but the two things that come to mind are 1) failure in the plasmid prep stage or 2) contaminant in the experimental samples such that your colonies are not actually E. coli with a plasmid.
Dear Prof Michael J. Benedik ,
Thank you a lot for your comment.
I am going to check whether these two possibilities might be the case.
Regards,
Albus.
Going for a 9-fragment assembly in Golden Gate cloning is pushing it a bit too much. The larger the number of fragments, the higher the probability that something will go wrong. The following might help:
Preprint Optimization of Golden Gate assembly through application of ...
That said,
- Have you checked that the Bsa I being used is actually working?
- Have you checked the T4 ligase?
Hope it helps!
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