Competent cells

1uL Ligase

5uL Buffer

2uL Pet28a vector

3uL Insert

Transform the resulting 10uL

Cultivate in kanamycin + 100uL of IPTG


Bis-Tris 1M solution

Inoculate into sterilized vial and add around 50uL of liquid bacterial medium, depending on the tubidity

Stock solution of 10xPBS, used for the interlab

10ml solution

0.8g NaCl

0.02g KCl

0.144g Na2HPO4

0.024g KH2PO4

0.0133g CaCl2-2H2O

0.01g MgCl2-6H2O

Plasmid digestion

With the samples in ice, add MiliQ water until reaching 16uL

Add 250ug of DNA (about 1uL if the concentration is high)

2.5 uL NEB buffer 2

0.5uL BSA

0.5uL ECOri

0.5uL Pstl

Spin the sample and maintain at 37ºC for 30 minutes

When running the electrophoresis:

10% gel (i.e. 0.3g of agarose to 30ml of TAE)

Run 5uL of ladder and 5uL of sample with the addition of 1uL of dye, if avaialable

Liquid LB medium

10g Bacto-triptona

5g yeast extract

10g NaCl

Add distilled water until reaching 1L

Adjust pH to 7.0

Optimum Medium

3g glucose

0.6g Na2HPO4

0.3g KH2PO4

0.05g NaCl

0.2g NH4Cl

20mL 1M Bis-Tris

1mL 10% Triton

100uL 1mg/mL thiamine

100uL 0.1M FeCl3-6H2O

100uL 1M MgSO4

Terrific Broth

14g Bacto-Triptone

24g yeast extract

4mk glycerol

100mL 0.17M KH2PO4

100mL 0.72M K2HPO4

TE 10X Buffer

100mL 1M Tris-HCl at pH 7.5~8

20mL 0.5M EDTA at pH 8

880mL H2O


0.5uL Sample

2uL Forward primer

2uL Reverse primer

10uL buffer



0.5uL Mg2Cl

0.5uL Phusion

31uL dH2O

All the other protocols were followed directly from the iGEM protocols