Hi everyone,
I am just trying to do Gibson assembly using HiFi DNA Assembly Master Mix with 4 inserts and a double digested vector with BsaI and BbsI. The information about inserts is as below:
1. 208 bps (17 bp overlap with vector)
2. 1254 bps (20 bp overlap with 1)
3. 815 bps (20 bp overlap with 2)
4. 114 bps (25 bp overlap with vector)
I used different molar ratios using NEB calculator:
I tried 1:2 for all
1:1 for fragment longer than 1000bps
1:5 for lower than 200 bps
and different combinations of all.
1 hour of incubation at 50 celsius. And also used both 10 µl and 20 µl reaction and finally transformed 5 µl of it to the competent cells.
but no colonies...
Would you please help me with it?
This will be tricky since you have a very 2 short fragments. I suggest you use 1:1 ratio for all except the short fragments use 1:10 ratio.
also be aware of the following:
1- gibson assembly should give a background of mis-constructs if you are unable to get any colonies it means your transformation is toxic. This is most likely due to using 5ul to transform.
2- commercial neb-10 indicates a max of 2ul gibson rxn per 50ul of competent cells. So try your old assemblies with this amount and see if it works.
3-For other strains I suggest you dilute your reaction before transforming but since this is a difficult assembly, an alternative is ethanol precipitate the rxn and transform all of it.
4- You can PCR your GA rxn to get a single gibson fragment by using the assembly primer for the edge fragments hence you will greatly simplify your reaction since it become a single fragment gibson reaction.
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