Thu, Oct 07 21

Gibson assembly transformtions #

Picked up plates set up from yesterday’s transformations to find disappointment. Very few colonies grew with some colonies having no successful transformations.

Colony counts #


At least the control is negative. Not great growth overall though. I did have to use regular LB instead of recovery medium but I’m not sure that would be the main factor here. It did not seem to matter which backbone was used in the Gibson assembly reactions. Saved some master mix from yesterday. Could also be that cells were not great in this round. Gibson assembly reactions were basically the same as what I did previously with VR 8, 12, 15 and got colonies for all of those samples.

At 2:30 PM I used sterile pipette tips to transfer colonies to individual glass tubes containing 3 ul of LB + 100 ug / ml AMP and placed onto shaker in 37C hotroom at 200 rpm to grow overnight. I plated colonies from samples VR 12, 15, 16, 25, 23 and 21.

Transformations of electro-competent cells #

Since it could be possible that cells used in last transformation were bad transforming into electro-competent cells following the lab protocol for electro-poration of E. coli and using the Gibson reactions from 10/6/21. Transformation conditions are below. Only samples that did not successfully transform overnight (17, 20, 27, and 31) were transformed.

  • 4 ul Gibson assembly reaction mix per sample
  • 20 ul of electro-competent cells instead of 25 ul as is easier to pipette without bubbles using the p20.

After added transformation mix I used pre-set E. coli electroporation protocol and all samples arced. I then repeated electroporation protocol with new cells but only using 0.5 ul of Gibson assembly reaction per sample. This time only VR-31 arced. I transferred the successfully shocked samples to 500 ul LB in 14 ml falcon tubes and places in 37C room for 1/2 to recover. I then redid electroporation with new VR-31 sample using 40 ul H20 and 0.5 ul Gibson reaction. Electroporation was successful this time. I then followed same recovery protocol as other samples. All samples transferer / handled under flame with filter tips.

After recovery of all samples plated VR 17, 20, 27 and 31 using 2 samples per plate with either 150 or 200 ul of transformed media. Placed all samples in the hot room to incubate overnight at 37C.