Hiya,

hope you're well and thank you for your time,

We've been trying to construct plasmids using Golden Gate but there are many issues and we don't know how to further progress our project.

1. We transform the cells using electroporation (we are unable to get chemical transformation to work)

2. We miniprep the cells and have big issues

NEB miniprep kit:

We've ranged between 1.5-5ml of culture, OD600 ranged between 0.7-1.7

The avg DNA conc is ~81ng/ul with the A260/280 and A260/A230 ratios being 1.7 and 1.2

We are unsure why the A260/A230 ratio is so low. It is often 0.6.

Beckman miniprep kit:

Conc is avg 392 with the ratios being 2.2 and 2.3

We are unsure why the A260/A280 ratio is so high. Could it be RNA contamination?

3. We run a gel of the miniprepped plasmids (digested) and no bands come up

We do a digestion and then we run it on a 1% agarose gel. We expect bands at 1800kbp and then depending on the plasmid, between 600-2000bps but there are no bands on the gel except wide bands smaller than 200bp

4. We golden gate plasmids

We do not have a positive control

We run the golden gate plasmids on a gel and they also do not show up.

Any advice would be so greatly appreciated, Thank you so much!