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               <h2>PDF NOTEBOOK</h2>
 
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                 <a href="https://static.igem.org/mediawiki/2018/f/f2/T--USP-Brazil--Notebook.pdf">Notebook.pdf</a>

Latest revision as of 20:22, 16 October 2018

Wiki - iGEM Brazil

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:

Primer Forward: 5’-TGATTACTGAATTCGCGGCCGCTTCTAG-3’

Primer Reverse: 5’-TGATTACTCTGCAGCGGCCGCTACTAGTA-3’

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

PDF NOTEBOOK

Notebook.pdf