Sequencing Results from Lac promoter/silk/spycatcher in psb1c3

Results looked mostly good with two small problem areas. I will be ordering a primer that starts upstream of this area to check by sequencing again.

Transformation with BBa_K525998 and Silk

With electrocompetent cells:

Thaw a 50uL aliquot of electro-competent E. coli cells on ice for 10 minutes. Chill transformation cuvettes on ice. Warm up SOC at 37C.
Add 2uL diluted (1:100) vector+insert to 50uL competent cell stock.
Add stock in between the plates of the electroporation cuvette.
Take out warmed SOC and prepare incubation tubes.
Place cuvette in pulse machine
1. Setting: 1.8 kV
2. Preload 950uL of SOC.
Press pulse and immediately add 950uL SOC to cuvette.
Pipet up and down two times, then add to incubation tube.
Incubate at 37C for one hour.
1. While waiting, warm up two chloramphenicol plates (1:1, 1:10)
2. After incubation, pellet the bacteria and remove supernatent. Add 101 uL of SOC.
Make a 1:10 dilution in SOC .
Plate 100uL of each sample and spread with beads.
Incubate at 37C (incubated at 4:30PM)

Last time I used electrocompetent cells, I got only 1 colony. As a backup I will make another plate using BL21 chemically competent cells at a 1:10 dilution.

With BL21:

I picked 8 colonies off the 1:10 plate and suspended each in 100uL Nuclease Free Water.

using Q5, transformed E. coli, and the VF2 and VR primers in preparation for insertion into psb1c3
Two reactions, one for each of the colonies.


Component	Volume (out of 25uL)
5X Q5 Reaction Buffer	5uL
10mM dNTPS	0.5uL
10mM primer 10	1.25uL
10mM primer 11	1.25uL
Transformed cells in ddH2O	1uL
Q5 High Fidelity DNA Polymerase	0.25uL
Nuclease Free Water	15.75uL