Paul.xu at 00:03, 23 March 2018

2018-03-23T00:03:03Z

New page

== 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

[[Category:Antibodies]]

=== 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

Phage Display - Revision history

Paul.xu at 00:03, 23 March 2018