REAGENTS
Lysozyme from chicken egg white (Sigma-Aldrich, cat. no. L6876)
Deoxyribonuclease I from bovine pancreas (DNaseI) (Sigma-Aldrich, cat. no. L6876)
T4 DNA ligase (Promega, cat no. M180A)
Phusion DNA Polymerase (Thermo Fischer Scientific, cat. no. F530S)
KAPA Taq PCR kit (Sigma-Aldrich, cat. no. KK1014)
Growth media and agar plates
Bacto tryptone (Duchefa, cat. no. T1332)
Glucose (Sigma-Aldrich, cat. no. G8270)
LB medium, high salt (Duchefa, cat. no. L1704)
Micro agar (Duchefa, cat. no. M1002)
Yeast extract (Duchefa, cat. no. Y1333)
Chemicals
Sodium chloride (Carl Roth, cat no. 3957)
Glycerol (VWR, cat. no. 24387.292)
IPTG, dioxane-free (Thermo Fischer Scientific, cat. no. R0392)
Trizma (Tris) base (Sigma-Aldrich, cat. no. T1503)
Restriction enzymes
SalI (New England BioLabs, cat. no. R0138)
SapI (New England BioLabs, cat. no. R0569)
DNA purification kits
QIAprep Spin Miniprep Kit (QIAGEN, cat. no. 27104)
Wizard® SV Gel and PCR Clean-Up System (Promega, cat. no. A9281)
Antibiotics
Ampicillin sodium salt (Carl Roth, cat. no. K029.2)
Protease Inhibitors
AEBSF (Carl Roth, cat. no. 2931.3)
Leupeptin hemisulphate (Carl Roth, cat. no. CN33.4)
Columns
Superdex™ 200 10/300 GL (Cytiva (GE Healthcare), cat. no. 17517501)
HisTrap FF 5 ml (Cytiva/GE Healthcare, cat. no. 17525501)
Primers
TU89*: 5’-CCTTGAGCTCTTCGTCCCTGAGACTCTCCTG-3’
TU92: 5’-ATCCATGAAGCCTATCAG-3’
TU93: 5’-AGATGTACCCTAAACTGG-3’
EP230: 5’-AGGACTGCTCTTCCACTGGAGACGGTGACCTGGGT-3’
TU32: 5’- GAGAAAATACCGCATCAGGC-3’
GIII: 5′-CCACAGACAGCCCTCATAG-3′.
* The 3'-sequence of primer TU89 (TCCCTGAGACTCTCCTG) is the complement to the ORF encoding residues 18 to 23 (SLRLSC) of the consensus sequence of a nanobody (IMGT numbering). If the sequence of the parental nanobody is different in this conserved region, one can adapt the TU89 primer accordingly to maintain the sequence of the parental nanobody in the megabody.
REAGENTS SETUP
LB-ampicillin glucose plates. Dissolve 25 g of LB medium (high salt) and 15 g of agar in 900 ml of ddH2O. Autoclave the mixture and cool it to 50 °C. Supplement it with 100 ml of 20% (wt/vol) glucose and 1 ml of 100 mg/ml ampicillin (0.20-μm-filter–sterilized) and pour it into plates. The plates can be stored for one month at 4 °C.
TB medium. Dissolve 12 g of Bacto tryptone, 2.3 g of KH2PO4, 12.5 g of K2HPO4, 24 g of yeast extract and 2.5 ml of glycerol in 1 liter of ddH2O. Autoclave the medium and store it for up to six months at 4 °C or for one month at RT.
PE buffer. 150 mM NaCl, 1 mM EDTA, 50 mM Tris pH 8, and 20% w/v sucrose, 0.5 mg/ml lysozyme, 0.12 mg/ml AEBSF and 0.5 ug/mL leupeptin hemisulphate. Preparation: add lysozyme, AEBSF and leupeptin hemisulphate just before use.
Washing buffer: 500 mM NaCl, 100 mM Tris pH 7.4,
Elution buffer: 500 mM NaCl, 100 mM Tris, 500 mM imidazole pH 7.4
Gel filtration (GF) buffer: 140 mM NaCl, 10 mM Tris-HCl pH 7.4