Phage Display

Reagents

M13K07 from 

M13K07 helper phage amplification

Infect 200 mg/mL XLI-Blue or TG1 at an OD600 of 0.4 with 10 mL of 100-fold serial dilutions of M13KO7 helper phage in a 37 ℃ water bath for 30 min. Add to 3 mL molten top agar (50 ℃) and pour onto warm LB plates. Allow to set and incubate overnight at 37 ℃.

Pick a small plaque into 5 mL of fresh SB at an OD600 of 0.4. Grow for about 2hr shaking at 37 ℃.

Add to 500 mL SB in a 2 litre flask and grow shaking at 37 ℃ for 1hr. Add kanamycin to a final concentration of 50 mg/mL. Grow overnight shaking at 37 ℃.

Spin overnight culture at 10,800 g for 15 min.

Add 100 mL 5 X PEG/NaCl to 400 mL supernatant and leave for 1 hr on ice.

Spin 10,800 g for 30 min. Pour away PEG/NaCl.

Re-suspend the pellet in 8 mL PBS and spin at 11,600 g for 10 min to remove any remaining bacterial debris (the titer of helper phage should be in the range of 1012-1013 pfu/mL).

Preparation of bacterial cells

1. Pick a single colony from a freshly streaked plate with the phagemid-containing bacteria and inoculate a 3-ml culture of 2xYT medium supplemented with ampicillin 100 µg/ml and glucose 1% w/v. In absence of IPTG and presence of glucose 1% w/v, the expression of the g3p fusion protein is repressed; this helps preventing loss of phagemid or selection of insertless clones, and improves library integrity.

2. Incubate o/n at 37ºC with agitation at 250 rpm.

3. In the morning, dilute an aliquot of the culture 1:20 v/v with fresh 2xYT medium in a new culture tube and incubate for one hour at 37ºC with agitation at 250 rpm. It is important to dilute the glucose to 0.1% or less to prevent the catabolite repression of the lac operator. 4. Take a small aliquot in sterile conditions and measure the absorbance at 600 nm of a 1:10 dilution in 2xYT medium (A600). With SS320 or TG1 strains, absorbance should be close to 0.5 OD and cells are ready to be transduced. If necessary adjust the length of the incubation to your conditions

Helper phage transduction 

It is recommended that incubate directly at 37ºC with agitation at 250 rpm for 30 min to one hour after adding the helper phage. The number of transductants increases over time, especially during the last 30 min of incubation. The total number of transductants is not increased by either pre-incubating bacteria without agitation for 10 min prior to adding the helper phage (sometimes said to regenerate pili destroyed by strong agitation) or after adding the phage (sometimes said to promote transduction). In fact we have noted that in our conditions a lack of agitation during the transduction period results in smaller number of transductants and variable virion production. Add  helper phage when the bacterial culture reaches an optical density at 600 nm between 0.4 OD and 0.5 OD; large amounts of nonsuperinfected cells may decrease virion production above 0.5 OD while disparities caused by differences in phage growth rates will be amplified at lower ODs.

1. For product PH010L (concentrated M13KO7), add 1 µl of M13KO7 phage per 1 ml of bacterial culture with A600 at 0.5 OD. This corresponds to a multiplicity of infection (MOI) around 18 and a number of pfu roughly 3 times the number of bacteria in the culture medium. For product PH010S, use 9 μl of M13KO7 phage per 1 ml of bacterial culture with A600 at 0.5 OD. This corresponds to a MOI around 9 and a number of pfu equivalent to 1.5 time number of bacteria in the culture medium.

2. Incubate for 1 h at 37°C and 250 rpm.

3. Add ampicillin 100 µg/ml, kanamycin 50 µg/ml, and IPTG 200 µM; lower the temperature to 30ºC and incubate 8 h to overnight before proceeding to phage purification. The amount of IPTG to add depends on the phagemid, e.g. phagemids such as pADL-23c, pHEN2 or pComb3 do not require IPTG while pADL-10b does require IPTG

Primer Design

Mouse Primers ===

3' LC  primer

Ck1 CATGTCTAGAACACTCATTCCTGTTGAAGCTCTTG

5' LC primers

VkA CATGGAGCTCGATGTTTTGATGACCCAAACTCCA
VkB GATCGAGCTCGACATTGTGCTCACCCAATCTCC
VkC CATGGAGCTCGACATTGTGCTRACCCAGTCTTCCA
VkD CATGGAGCTCGACATCCAGATGACNCAGTCTCAA
VkE CATGGAGCTCCAAATTGTTCTCACCCAGTCTCCA
VkF CATGGAGCTCGAAAATGTGCTTCACCCAGTCTCCA

3' HC primers

IgG1 AGGCTTACTAGTTATGCAAGGCTTACAACC
IgG2A AGGCTTACTAGTACAGGGCTTGATTGTGGGCCC
IgG2B AGGCTTACTAGTACAGGGGTTCAGTGTTGAAATGG

5' HC primers

IA  TGGAGGCTTCTCGAGGAKGTGCAGCTTCAGGAGTC
IB  TGGAGGCTTCTCGAGCAGGTGCAGCTGAAGSAGTC
IIA  TGGAGGCTTCTCGAGSAGGTCCAGCTGCARCAGTC
IIB  TGGAGGCTTCTCGAGCAGGTCCARCTGCAGCAGYTTGG
IIC  TGG AGGCTTCTCGAGGAGGTTCAGCTGCAGCAGTC
IIIA  TGGAGGCTTCTCGAGGARGTGAAGCTGGTGGARTCTGG
IIIB  TGGAGGCTTCTCGAGGAGGTGAAGCTTCTGGAGTCTGG
IIIC  TGGAGGCTTCTCGAGGAAGTGAAGCTTGAGGAGWCTGG
IIIDA  TGGAGGCTTCTCGAGGAAGTGCAGCTGGTGGAGTCTGG
IIIDB  TGGAGGCTTCTCGAGGAAGTGATGCTGGTGGAGTCTGG
VA  TGGAGGCTTCTCGAGGAGGTYCAGCTKCAGCAG
CH1  GCCAAAACGACACCCCCA

Human Primers

3' LC kappa primer

Ck Ck1z GCGCCGTCTAGAATTAACACTCTCCCCTGTTGAAGCT
CTTTGTGACGGGCGAACTCAG

5' LCkappa primers

1 Vk1a GACATCGAGCTCACCCAGTCTCCA
 2 Vk2a GATATTGAGCTCACTCAGTCTCCA
3 Vk3a GAAATTGAGCTCACGCAGTCTCCA

kappa Constant CONk ACTGTGGCTGCACCATCTG

3' LC lambda primer

Clambda Cl2 CGCCGTCTAGAACTATGAACATTCAGG

5' LC lambda primers

1,2    Vl1,2        CAGTCTGAGCTCACTCAGCCRCCC
 3       Vl3          CAGCCTGAGCTCACTCAG
4,5,9   Vl4,5,9    TCTGTGGAGCTCCAGCCGCCCTCAGTG
6       Vl6 AATTTTGAGCTCACTCAGCCC
7       Vl7 CAGGCTGAGCTCACTCAGGAG
8       Vl8 CAGACTGAGCTCACCCAGGAG
10     Vl10 CAGGCAGAGCTCACTCAGCCA
lambda Constant CONl2 AAGGCTGCCCCCACGGTCACTCTG

Human HC Primers 3' HC primers

g: gamma

Cg1z    g1 GCATGTACTAGTTTTGTCACAAGATTTGGG
 Cg2z   g2 CGGTGGACTAGTGACACAACATTTGCG
Cg3z   g3 TGGGCAACTAGTGCATGTGTGAGTTGTG
Cg4z   g4 TGGGCAACTAGTGCATGGGGGACCATATTTGGA
CONga   g1,2,3,4   TCCACCAAGGGCCCATCG

5' HC primers

VH1a   1 and 4   CAGGTGCAGCTCGAGCAGTCTGGG
 VH1f   1 and 4   CAGGTGCAGCTGCTCGAGTCTGG
VH2fN   2         CAGATCACCCTCGAGGAGTCTGGT
VH3a    3         GAGGTGCAGCTCGAGGAGTCTGGG
VH3f     3         GAGGTGCAGCTGCTCGAGTCTGGG
VH5f    5          GAGGTGCAGCTCGAGCAGTCTGGA
VH6f    6          CAGGTACAGCTGCTCGAGTCAGGTCCA

AY Protocol

p19BsrG1
AGCTGTCATGTACACTCCCAAGTTCAACTTCAGCAGTCTGGCGC
pPaniBsrG1
AGCTGTCATGTACACTCCCAAGTTCAACTGCAAGAGTCTGGCCCT
pHer2BsrG1
AGCTGTCATGTACACTCCGAAGTTCAACTTGTGGAATCTGGCGGC
pCMVFor
CGCAAATGGGCGGTAGGcGTG
pCMVrev
CAACATAGTTAAGAATACCAGTC
VHH1SfiFor
CATGCCATGACTCGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGGTGCAGTCTGG
VHH2SfiFor
CATGCCATGACTCGCGGCCCAGCCGGCCATGGCCCAGGTCACCTTGAAGGAGTCTGG
VHH3SfiFor
CATGCCATGACTCGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGGTGGAGTCTGG
VHH4SfiFor
CATGCCATGACTCGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGCAGGAGTCGGG
pPaniBsrG1
AGCTGTCATGTACACTCCCAAGTTCAACTGCAAGAGTCTGGCCCTG
p19BsrG1
AGCTGTCATGTACACTCCCAAGTTCAACTTCAGCAGTCTGGCGC
pHer2BsrG1
AGCTGTCATGTACACTCCGAAGTTCAACTTGTGGAATCTGGCGGC
HCkRev 
AGACTCTCCCCTGTTGAAGCTCTT
pADLFor
TTTACACTTTATGCTTCCGGCTCGTATGTTG
pADLRev
AGGTTTTGCTAAACAACTTTCAAC
pG2CdBssHII
GCCAGGCTCAAGGCCCGCATGCCCGACGGCGA
HuIgMRev
TGGAAGAGGCACGTTCTTTTCTTT
C_Rev
AGACTCTCCCCTGTTGAAGCTAAG
C_Rev
TGAAGATTCTGTAGGGGCCACTGTCTT
HuJH12Rev
GGAAGATCTAGAGGAACCACCTGAGGAGACGGTGACCAGGGTGCC
HuJH3Rev
GGAAGATCTAGAGGAACCACCTGAAGAGACGGTGACCATTGTCCC
HuJH45Rev
GGAAGATCTAGAGGAACCACCTGAGGAGACGGTGACCAGGGTTCC
HuJH6Rev
GGAAGATCTAGAGGAACCACCTGAGGAGACGGTGACCGTGGTCCC
pCHIBack
GCTCGGGGAAGTAGTCCTTCACCAGG
pCKBACK
TTCCAGATTTCAACTGCTCATCAG
HuVH1B7AFor
TACTCGCGGCCCAGCCGGCCATGGCTCAGRTGCAGCTGGTGCARTCTGG
HuVH1CFor
TACTCGCGGCCCAGCCGGCCATGGCTSAGGTCCAGCTGGTRCAGTCTGG
HuVH2BFor
TACTCGCGGCCCAGCCGGCCATGGCTCAGRTCACCTTGAAGGAGTCTGG
HuVH3BFor
TACTCGCGGCCCAGCCGGCCATGGCTSAGGTGCAGCTGGTGGAGTCTGG
HuVH3CFor
TACTCGCGGCCCAGCCGGCCATGGCTGAGGTGCAGCTGGTGGAGWCYGG
HuVH4BFor
TACTCGCGGCCCAGCCGGCCATGGCTCAGGTGCAGCTACAGCAGTGGGG
HuVH4CFor
TACTCGCGGCCCAGCCGGCCATGGCTCAGSTGCAGCTGCAGGAGTCSGG
HuVH5BFor
TACTCGCGGCCCAGCCGGCCATGGCTGARGTGCAGCTGGTGCAGTCTGG
HuVH6AFor
TACTCGCGGCCCAGCCGGCCATGGCTCAGGTACAGCTGCAGCAGTCAGG
HuV_5For
GGTGGCGGTGGCTCGGGCGGTGGTGGGGAAACGACACTCACGCAGTCTCC
HuV_6For
GGTGGCGGTGGCTCGGGCGGTGGTGGGGAAATTGTGCTGACTCAGTCTCC
HuV_1AFor
GGTGGCGGTGGCTCGGGCGGTGGTGGGCAGTCTGTGCTGACTCAGCCACC
HuV_1BFor
GGTGGCGGTGGCTCGGGCGGTGGTGGGCAGTCTGTGYTGACGCAGCCGCC
 
HuV_2For
GGTGGCGGTGGCTCGGGCGGTGGTGGGCARTCTGCCCTGACTCAGCCT
HuV_3AFor
GGTGGCGGTGGCTCGGGCGGTGGTGGGTCCTATGWGCTGACTCAGCCACC
HuV_3BFor
GGTGGCGGTGGCTCGGGCGGTGGTGGGTCTTCTGAGCTGACTCAGGACCC
HuV_4For
GGTGGCGGTGGCTCGGGCGGTGGTGGGCACGTTATACTGACTCAACCGCC
HuV_5For
GGTGGCGGTGGCTCGGGCGGTGGTGGGCAGGCTGTGCTGACTCAGCCGTC
HuV_6For
GGTGGCGGTGGCTCGGGCGGTGGTGGGAATTTTATGCTGACTCAGCCCCA
HuV_78For
GGTGGCGGTGGCTCGGGCGGTGGTGGGCAGRCTGTGGTGACYCAGGAGCC

HuV_9For
GGTGGCGGTGGCTCGGGCGGTGGTGGGCWGCCTGTGCTGACTCAGCCMCC
LinkerFor, verified
GGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGGCTCGGGCGGTGGTGGG

HuJ_1RevSfiI, verified
TGGTGTTGGCCTCCCGGGCCACTAGTACGTTTGATHTCCASYTTGGTCCC
HuJ_1_2RevSfiI, verified
TGGTGTTGGCCTCCCGGGCCACTAGTACCTAGGACGGTGACCTTGGTCCC

HuJ_2RevSfiI, verified
TGGTGTTGGCCTCCCGGGCCACTAGTACGTTTAATCTCCAGTCGTGTCCC

HuJ_2_2RevSfiI, verified
TGGTGTTGGCCTCCCGGGCCACTAGTACCTAGGACGGTCAGCTTGGTCCC

HuJ_3RevSfiI, verified
TGGTGTTGGCCTCCCGGGCCACTAGTACYTAAAACGGTGAGCTGGGTCCC