Notebook
Circuit construction
To build up all the 12 plasmids, we mostly used biobricks from the 2017 distribution kit. The flowchart below shows all the cloning procedures we have planned.
Protocols
The circuit construction was done following these protocols:
Heat shock transformation
After thawing the cells on ice for 10 minutes, add 1-5uL of plasmid to 50uL of competent cells and mix gently. Incubate the cells on ice for 20-13 minutes and spread 20uL of antibiotics for each plate. Afterwards, waterbath the cells at 42°C for 1 minute. Put the tube back on ice for 2 minutes. Add 950uL of LB to the tube and incubate at 37°C for 30-60 minutes. Centrifuge and ressuspend the cells with 100uL LB. Plate and incubate the cells ovvernight at 37°C.
PCR Protocol
| DNA | Forward primer 10mM | Reverse primer 10mM | dNTPs 10mM | 10x Buffer | MgCl2 50mM | Taq | H2O |
|---|---|---|---|---|---|---|---|
| 1uL | 0,5uL | 0,5uL | 0,5uL | 2,5uL | 0,75uL | 0,1uL | to 25uL |
We have ordered from Exxtend the following primers for PCR of BioBricks. They are complementary to the BioBricks prefix and suffix, and have a 5' overhang for the ocasion of wanting to amplify a DNA fragment for cloning, as the proximity of the EcoRI and PstI restriction sites to the end of the fragment would hinder the enzymes activity:
Cycles for the pcr were as following:
| Step 1 | Step 2 | Step 3 | Step 4 | Step 5 | Step 6 | Step 7 |
|---|---|---|---|---|---|---|
| 95ºC 5min | 95ºC 30s | 60ºC 30s | 72ºC 1min/Kb | Return to Step 2 30x | 72ºC 5min | 4ºC |
Digestion Protocol
We used EcoRI, SpeI, NotI, XbaI or PstI for digestion of biobricks. Depending on the use we were going to make of the parts, we used different quantities of DNA for digestion (to obtain a linearized vector for 3A assembly, for example, we used less DNA to minimize the amount of background, empty vector transformations)
| Plasmid | 10x 2.1 Buffer | Enzymes | H2O |
|---|---|---|---|
| 15uL | 2uL | 1uL each | To 20uL |
Incubate the restriction digest at 37°C for 30 minutes and then 80°C for 20 minutes, for heat-inactivation in case of a subsequent 3A assembly. If the fragments were to be used in conventional ligation, we purified it from an agarose gel, loading all of the digestion.
Ligation Protocol
| Vector | Insert | 10x Ligase Buffer | T4 Ligase | H2O | |
|---|---|---|---|---|---|
| Stdt. Ligation | 1,5uL | 10uL | 2uL | 1uL | To 20uL |
| 3A assembly | 2,3uL | 2,3uL each | 1uL | 1uL |
Ligate 16°C for 60 minutes and heat-inactivate at 80°C for 20 minutes