The cDNA used as template of my inserts was the result of pooling the same volume of each V gene PCR reaction. Considering the PCR efficiency the same for each reaction, my V gene inserts were resembling the same relative expression of VH and VL families as in the B cells infiltrating the brain. The PCR products were a reliable copy of the humoral immune response of the patients and I cloned the amplicons by digesting with NcoI and XhoI (for the heavy
75 chains) and NotI and SalI (for the light chains) overnight (O.N.) at 37°C followed by a boost of 1 µl of XhoI/NcoI or NotI/SalI for 2 hours as in the reaction detailed below: Recipe µl Insert 30 XhoI 2 NcoI 2 10x Buffer 10 Water 55 BSA 1 Total volume 100
Digested PCR products were checked for presence of a single band, corresponding to approximately 400 bp, on 1% agarose gel to determine quantity and quality. The remaining volume was then run on a 0.8% low melting point preparative agarose gel and the band excised with a sterile blade being visualized by a UV transilluminator. The DNA was extracted using the QIAquick Gel extraction kit (Qiagen Ltd., UK).
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2.6.2.1 Preparation of Vector
The bacterial growth media formulations were prepared as detailed in table 2.8. 2TY broth and TYE agar were prepared in double-distilled water and the pH adjusted to 7.4. The minimal media components were autoclaved separately except for the glucose and thiamine that were sterile filtered.
2TY broth
Tryptone 16 g/l Biogene Ltd, Kimbolton, Cambs, UK Yeast Extract 10 g/l Biogene Ltd.
Sodium Chloride 5 g/l BDH Laboratory Supplies (BDH), supplied by VWR International Ltd, Lutterworth, UK TYE agar
Tryptone 10 g/l Biogene Ltd.
Yeast Extract 5 g/l Biogene Ltd.
Sodium Chloride 8 g/l BDH Bacto-Agar 15 g/l Biogene Ltd. Minimal Salt (2x M9) Na2HPO4 12g/l NA KH2PO4 6g/l NA NaCl 1g/l NA NH4Cl 2g/l NA Minimal Media 2x M9 500 ml NA 3% Agar 500 ml NA 20% Glucose 20 ml NA MgSO4 1M 2 ml NA CaCl2 1M 0.1 ml NA Thiamine (10 mg/ml) 1 ml NA
Table 2.8 Bacterial growth media formulations
The plasmid bacterial stock was cultured at 37°C O.N. in 10 ml 2xTY, 100µg/ml ampicillin, 1% glucose. Plasmid DNA was isolated using the Qiagen Plasmid Midiprep kit (Qiagen Ltd., UK). The plasmid was digested with NcoI and XhoI as detailed previously and run on a 0.8% low melting point agarose gel. The digested vector ~ 5Kb was excised out of the gel as for the inserts and extracted using the QIAquick Gel extraction kit (Qiagen Ltd., UK). An amount of 10 µl of DNA was run on a 1% agarose gel to determine quantity and purity.
The resulting DNA was purified by precipitation at -20°C for 15-20 min in a mixture of water, NH4 acetate and 3x volume of ethanol. After the ethanol
precipitation the sample was centrifuged at 4°C at max speed for 15 min. The pellet was washed with increasing concentrations of ethanol, left to dry and resuspended in water.
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2.6.2.2 Ligation
To optimize the ratio for the ligation reaction, test ligations were performed in different ratios of insert to vector (1:1, 3:1, 5:1 and 10:1). To estimate the correct concentration of vector and insert, the preparations were run on agarose gel and quantified on an UV transilluminator. The desired ratio insert:vector was obtained adding the appropriate volumes to the test ligation reaction mixture in a total volume of 20 µl as reported below. Ligation reactions were incubated O.N. at 37°C. Large scale ligations were concentrated by ethanol precipitation.
Recipe µl Insert x Vector x Buffer 2 Water x T4 ligase 1 Total volume 20
2.6.2.3 Preparation of competent E. coli TG1
E. coli, TG1 strain, cells were cultured O.N. shaking in 2xTY medium and
incubated at 37°C. The cultured bacteria were inoculated in baffled flasks and grown in a 37°C shaker, cooled on ice for 10 min before transferring to cold centrifuge bottles. The chilled culture was spun for 15 min at ~ 4500 rpm and the resulting pellet washed with multiple passages in HEPES solution. In the final step the pellet was resuspended in 10% glycerol. The cells were frozen as 100µl aliquots at -80°C or kept on ice for fresh use.
2.6.2.4 Transformation
The electrocompetent bacteria were thawed if necessary and mixed with 4 µl of ligated phagemid in a 0.2 cm electroporation cuvette. The cuvette with the mixture was placed in the electroporator Biorad MicroPulser (Bio-Rad Laboratories Ltd., UK) and pulsed following the manufacturer’s instructions. The electroporated cultures were plated and incubated at 37°C O.N.. The plates were scraped and the library stock stored at -80°C.
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2.6.2.5 Library size
The evaluation of the MS libraries size was obtained by plating serial dilutions of the libraries on agar plates containing ampicillin for colony counting.
2.6.2.6 Sequencing
Sanger sequencing
A small portion of the PCR products were cycle sequenced using the BigDye chemistry material kindly supplied by Dr Alex Pearson and then sent for sequencing to The Institute of Cancer Research, London, UK. The PCR product templates were prepared by half-reactions with 1l PCR template per 20µl total volume, and the extension reaction performed by thermal cycler according to the following schedule:
Denaturation 3 minutes 96C 1x Denaturation 30 seconds 96C
25x Annealing 15 seconds 50C
Extension 4 minutes 60C
The remaining PCR products were sent for sequencing directly to The Genome Centre (WHRI, QMUL) and sequenced by BigDye 3.1 chemistry with visualization on the ABI 3730xl capillary sequencer.
The following primers were used in sequencing of the heavy and light chains of scFv from the MS antibody phage display libraries:
PHEN: 5’_CTATGCGGCCCCATTCA_3’
LMB3: 5’_CAGGAAACAGCTATGAC_3’