VCSM13 is a derivative of M13KO7 originally commercialized by Stratagene and often used in phagedisplay; the phenotype of VCSM13 has never been published and exact sequence is unclear.To propagate (amplify) the VCSM13 helper phage requires using the E. coli TG1，ER2738 or strainXL1-Blue MRF′ ，because those strain harbors the supE44 mutation, which provides a glutamine suppressor tRNA.The VCSM13 helper phage is used strictly to produce single stranded DNA from the already excised phagemid. (This is also what M13K07 is used for.)
Storing the Helper Phage
Recommends storing them at -80℃ with 50% Glycerol. As long as there is a -80℃ stock in the lab, the amplified lab prep can be stored at+4℃.
Titering the Helper Phage
1. Streak out TG1 onto an 2YT plate. Pick a single colony of TG1, inoculate into 2YT and grow to OD600 = 0.6. Meanwhile prepare a serial dilution of the phage in PBS buffer. Expected number of phage should be about 1010pfu/ml; therefore dilute phage to create dilutions 10-4 to 10-7.
Note:
When preparing serial dilutions of bacteriophage, it is usually much more accurate to dilute 10μl into 100 to create a 10-1 dilution or 10μl into 1000μl to create a 10-2 dilution rather than to dilute 1 into 10μl or 1μl into 100μl because one tends to obtain grossly exaggerated titers resulting from small pipetting errors. This is particularly critical for the first dilution.
2.Add 200μl of TG1 cells at OD600 = 0.6 to individual sterile culture tubes (the number of phage dilutions you plan to plate) in a test tube rack.
3.Add 100μl of each serial dilution of helper phage to the culture tubes containing the TG1 cells.
4. Place the test tube rack into a 37℃ water bath for 15 minutes to allow the helper phage to attach to the cells.
5. Meanwhile melt NZY Top Agarose in the microwave and allow it to cool to ~48/50℃.
You need to pay close attention to the bottle containing the top agarose because it can quickly boil over; alternatively use a low power setting on the microwave.
6. At the conclusion of the 15-minute incubation, add ~3 ml of the NZY top agarose to the test tube containing the helper phage & E.coli. Remove the tube from the rack (which is still sitting in the water bath) and give it a quick flick of your wrist mixing the contents and immediately pour it onto an NZY plate. (I usually do three plates at one by pipetting 10 ml of the NZY top agarose from the bottle, dispensing ~3.3 to one tube, ~3.3 to second tube and then another ~3.3 to a third tube. I then pour the plates starting with the first tube I put the NZY into.)
7. Allow the top agarose to cool (~5 minutes).
8. Invert the plate and incubate overnight at 37℃.
9. Count the number of plaques.
Amplifying the Helper Phage
1. Streak out TG1 onto an 2YT plate.
2. Pick a single colony of TG1 and inoculate into 5ml 2×YT and grow until OD600 = 0.3 (~2.5×108 cells/ml).
3. Add helper phage at a multiplicity of infection (MOI) of 20:1 (phage-to-cells ratio). Do this for both VCSM13.
Note: (If amplifying VCSM13 helper phage, add kanamycin to a final concentration of 25μg/ml to the medium 30 minutes after the helper phage and cells have been allowed to grow together.)
4. Grow the culture at 37℃ with vigorous aeration (~300rpm) for 8 hours.
5. Heat the culture to 65℃ for 15 minutes.
6. Spin down the cell debris and transfer the supernatant to a fresh tube.
7. Titer the helper phage produced.
Vector sequence
1 aacgctacta ctattagtag aattgatgcc accttttcag ctcgcgcccc aaatgaaaat
61 atagctaaac aggttattga ccatttgcga aatgtatcta atggtcaaac taaatctact
121 cgttcgcaga attgggaatc aactgttaca tggaatgaaa cttccagaca ccgtacttta
181 gttgcatatt taaaacatgt tgagctacag caccagattc agcaattaag ctctaagcca
241 tccgcaaaaa tgacctctta tcaaaaggag caattaaagg tactctctaa tcctgacctg
301 ttggagtttg cttccggtct ggttcgcttt gaagctcgaa ttaaaacgcg atatttgaag
361 tctttcgggc ttcctcttaa tctttttgat gcaatccgct ttgcttctga ctataatagt
421 cagggtaaag acctgatttt tgatttatgg tcattctcgt tttctgaact gtttaaagca
481 tttgaggggg attcaatgaa tatttatgac gattccgcag tattggacgc tatccagtct
541 aaacatttta ctattacccc ctctggcaaa acttcttttg caaaagcctc tcgctatttt
601 ggtttttatc gtcgtctggt aaacgagggt tatgatagtg ttgctcttac tatgcctcgt
661 aattcctttt ggcgttatgt atctgcatta gttgaatgtg gtattcctaa atctcaactg
721 atgaatcttt ctacctgtaa taatgttgtt ccgttagttc gttttattaa cgtagatttt
781 tcttcccaac gtcctgactg gtataatgag ccagttctta aaatcgcata aggtaattca
841 caatgattaa agttgaaatt aaaccatctc aagcccaatt tactactcgt tctggtgttt
901 ctcgtcaggg caagccttat tcactgaatg agcagctttg ttacgttgat ttgggtaatg
961 aatatccggt tcttgtcaag attactcttg atgaaggtca gccagcctat gcgcctggtc
1021 tgtacaccgt tcatctgtcc tctttcaaag ttggtcagtt cggttccctt atgattgacc
1081 gtctgcgcct cgttccggct aagtaacatg gagcaggtcg cggatttcga cacaatttat
1141 caggcgatga tacaaatctc cgttgtactt tgtttcgcgc ttggtataat cgctgggggt
1201 caaagatgag tgttttagtg tattctttcg cctctttcgt tttaggttgg tgccttcgta
1261 gtggcattac gtattttacc cgtttaatgg aaacttcctc atgaaaaagt ctttagtcct
1321 caaagcctct gtagccgttg ctaccctcgt tccgatgctg tctttcgctg ctgagggtga
1381 cgatcccgca aaagcggcct ttaactccct gcaagcctca gcgaccgaat atatcggtta
1441 tgcgtgggcg atggttgttg tcattgtcgg cgcaactatc ggtatcaagc tgtttaagaa
1501 attcacctcg aaagcaagct gataaaccga tacaattaaa ggctcctttt ggagcctttt
1561 tttttggaga ttttcaacgt gaaaaaatta ttattcgcaa ttcctttagt tgttcctttc
1621 tattctcact ccgctgaaac tgttgaaagt tgtttagcaa aaccccatac agaaaattca
1681 tttactaacg tctggaaaga cgacaaaact ttagatcgtt acgctaacta tgagggttgt
1741 ctgtggaatg ctacaggcgt tgtagtttgt actggtgacg aaactcagtg ttacggtaca
1801 tgggttccta ttgggcttgc tatccctgaa aatgagggtg gtggctctga gggtggcggt
1861 tctgagggtg gcggttctga gggtggcggt actaaacctc ctgagtacgg tgatacacct
1921 attccgggct atacttatat caaccctctc gacggcactt atccgcctgg tactgagcaa
1981 aaccccgcta atcctaatcc ttctcttgag gagtctcagc ctcttaatac tttcatgttt
2041 cagaataata ggttccgaaa taggcagggg gcattaactg tttatacggg cactgttact
2101 caaggcactg accccgttaa aacttattac cagtacactc ctgtatcatc aaaagccatg
2161 tatgacgctt actggaacgg taaattcaga gactgcgctt tccattctgg ctttaatgag
2221 gatccattcg tttgtgaata tcaaggccaa tcgtctgacc tgcctcaacc tcctgtcaat
2281 gctggcggcg gctctggtgg tggttctggt ggcggctctg agggtggtgg ctctgagggt
2341 ggcggttctg agggtggcgg ctctgaggga ggcggttccg gtggtggctc tggttccggt
2401 gattttgatt atgaaaagat ggcaaacgct aataaggggg ctatgaccga aaatgccgat
2461 gaaaacgcgc tacagtctga cgctaaaggc aaacttgatt ctgtcgctac tgattacggt
2521 gctgctatcg atggtttcat tggtgacgtt tccggccttg ctaatggtaa tggtgctact
2581 ggtgattttg ctggctctaa ttcccaaatg gctcaagtcg gtgacggtga taattcacct
2641 ttaatgaata atttccgtca atatttacct tccctccctc aatcggttga atgtcgccct
2701 tttgtcttta gcgctggtaa accatatgaa ttttctattg attgtgacaa aataaactta
2761 ttccgtggtg tctttgcgtt tcttttatat gttgccacct ttatgtatgt attttctacg
2821 tttgctaaca tactgcgtaa taaggagtct taatcatgcc agttcttttg ggtattccgt
2881 tattattgcg tttcctcggt ttccttctgg taactttgtt cggctatctg cttacttttc
2941 ttaaaaaggg cttcggtaag atagctattg ctatttcatt gtttcttgct cttattattg
3001 ggcttaactc aattcttgtg ggttatctct ctgatattag cgctcaatta ccctctgact
3061 ttgttcaggg tgttcagtta attctcccgt ctaatgcgct tccctgtttt tatgttattc
3121 tctctgtaaa ggctgctatt ttcatttttg acgttaaaca aaaaatcgtt tcttatttgg
3181 attgggataa ataatatggc tgtttatttt gtaactggca aattaggctc tggaaagacg
3241 ctcgttagcg ttggtaagat tcaggataaa attgtagctg ggtgcaaaat agcaactaat
3301 cttgatttaa ggcttcaaaa cctcccgcaa gtcgggaggt tcgctaaaac gcctcgcgtt
3361 cttagaatac cggataagcc ttctatatct gatttgcttg ctattgggcg cggtaatgat
3421 tcctacgatg aaaataaaaa cggcttgctt gttctcgatg agtgcggtac ttggtttaat
3481 acccgttctt ggaatgataa ggaaagacag ccgattattg attggtttct acatgctcgt
3541 aaattaggat gggatattat ttttcttgtt caggacttat ctattgttga taaacaggcg
3601 cgttctgcat tagctgaaca tgttgtttat tgtcgtcgtc tggacagaat tactttacct
3661 tttgtcggta ctttatattc tcttattact ggctcgaaaa tgcctctgcc taaattacat
3721 gttggcgttg ttaaatatgg cgattctcaa ttaagcccta ctgttgagcg ttggctttat
3781 actggtaaga atttgtataa cgcatatgat actaaacagg ctttttctag taattatgat
3841 tccggtgttt attcttattt aacgccttat ttatcacacg gtcggtattt caaaccatta
3901 aatttaggtc agaagatgaa attaactaaa atatatttga aaaagttttc tcgcgttctt
3961 tgtcttgcga ttggatttgc atcagcattt acatatagtt atataaccca acctaagccg
4021 gaggttaaaa aggtagtctc tcagacctat gattttgata aattcactat tgactcttct
4081 cagcgtctta atctaagcta tcgctatgtt ttcaaggatt ctaagggaaa attaattaat
4141 agcgacgatt tacagaagca aggttattca ctcacatata ttgatttatg tactgtttcc
4201 attaaaaaag gtaattcaaa tgaaattgtt aaatgtaatt aattttgttt tcttgatgtt
4261 tgtttcatca tcttcttttg ctcaggtaat tgaaatgaat aattcgcctc tgcgcgattt
4321 tgtaacttgg tattcaaagc aatcaggcga atccgttatt gtttctcccg atgtaaaagg
4381 tactgttact gtatattcat ctgacgttaa acctgaaaat ctacgcaatt tctttatttc
4441 tgttttacgt gctaataatt ttgatatggt tggttcaatt ccttccataa ttcagaagta
4501 taatccaaac aatcaggatt atattgatga attgccatca tctgataatc aggaatatga
4561 tgataattcc gctccttctg gtggtttctt tgttccgcaa aatgataatg ttactcaaac
4621 ttttaaaatt aataacgttc gggcaaagga tttaatacga gttgtcgaat tgtttgtaaa
4681 gtctaatact tctaaatcct caaatgtatt atctattgac ggctctaatc tattagttgt
4741 tagtgcacct aaagatattt tagataacct tcctcaattc ctttctactg ttgatttgcc
4801 aactgaccag atattgattg agggtttgat atttgaggtt cagcaaggtg atgctttaga
4861 tttttcattt gctgctggct ctcagcgtgg cactgttgca ggcggtgtta atactgaccg
4921 cctcacctct gttttatctt ctgctggtgg ttcgttcggt atttttaatg gcgatgtttt
4981 agggctatca gttcgcgcat taaagactaa tagccattca aaaatattgt ctgtgccacg
5041 tattcttacg ctttcaggtc agaagggttc tatctctgtt ggccagaatg tcccttttat
5101 tactggtcgt gtgactggtg aatctgccaa tgtaaataat ccatttcaga cgattgagcg
5161 tcaaaatgta ggtatttcca tgagcgtttt tcctgttgca atggctggcg gtaatattgt
5221 tctggatatt accagcaagg ccgatagttt gagttcttct actcaggcaa gtgatgttat
5281 tactaatcaa agaagtattg ctacaacggt taatttgcgt gatggacaga ctcttttact
5341 cggtggcctc actgattata aaaacacttc tcaagattct ggcgtaccgt tcctgtctaa
5401 aatcccttta atcggcctcc tgtttagctc ccgctctgat tccaacgagg aaagcacgtt
5461 atacgtgctc gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
5521 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
5581 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
5641 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
5701 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
5761 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
5821 ctatctcggg acggatcgct tcatgtggca ggagaaaaaa ggctgcaccg gtgcgtcagc
5881 agaatatgtg atacaggata tattccgctt cctcgctcac tgactcgcta cgctcggtcg
5941 ttcgactgcg gcgagcggaa atggcttacg aacggggcgg agatttcctg gaagatgcca
6001 ggaagatact taacagggaa gtgagagggc cgcggcaaag ccgtttttcc ataggctccg
6061 cccccctgac aagcatcacg aaatctgacg ctcaaatcag tggtggcgaa acccgacagg
6121 actataaaga taccaggcgt ttccccctgg cggctccctc gtgcgctctc ctgttcctgc
6181 ctttcggttt accggtgtca ttccgctgtt atggccgcgt ttgtctcatt ccacgcctga
6241 cactcagttc cgggtaggca gttcgctcca agctggactg tatgcacgaa ccccccgttc
6301 agtccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gaaagacatg
6361 caaaagcacc actggcagca gccactggta attgatttag aggagttagt cttgaagtca
6421 tgcgccggtt aaggctaaac tgaaaggaca agttttggtg actgcgctcc tccaagccag
6481 ttacctcggt tcaaagagtt ggtagctcag agaaccttcg aaaaaccgcc ctgcaaggcg
6541 gttttttcgt tttcagagca agagattacg cgcagaccaa aacgatctca agaagatcat
6601 cttattaagg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
6661 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
6721 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
6781 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
6841 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
6901 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccgattc gagctcgccc
6961 cggggatcga ccagttggtg attttgaact tttgctttgc cacggaacgg tctgcgttgt
7021 cgggaagatg cgtgatctga tccttcaact cagcaaaagt tcgatttatt caacaaagcc
7081 gccgtcccgt caagtcagcg taatgctctg ccagtgttac aaccaattaa ccaattctga
7141 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
7201 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
7261 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
7321 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
7381 tgaatccggt gagaatggca aaagcttatg catttctttc cagacttgtt caacaggcca
7441 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
7501 cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
7561 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
7621 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
7681 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
7741 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
7801 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
7861 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
7921 cctcgagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat
7981 gtaagcagac agttttattg ttcatgatga tatattttta tcttgtgcaa tgtaacatca
8041 gagattttga gacacaacgt ggctttcccc ccccccccct gcaggtctcg ggctattctt
8101 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
8161 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aatttaaata tttgcttata
8221 caatcttcct gtttttgggg cttttctgat tatcaaccgg ggtacatatg attgacatgc
8281 tagttttacg attaccgttc atcgattctc ttgtttgctc cagactctca ggcaatgacc
8341 tgatagcctt tgtagacctc tcaaaaatag ctaccctctc cggcatgaat ttatcagcta
8401 gaacggttga atatcatatt gatggtgatt tgactgtctc cggcctttct cacccttttg
8461 aatctttacc tacacattac tcaggcattg catttaaaat atatgagggt tctaaaaatt
8521 tttatccttg cgttgaaata aaggcttctc ccgcaaaagt attacagggt cataatgttt
8581 ttggtacaac cgatttagct ttatgctctg aggctttatt gcttaatttt gctaattctt
8641 tgccttgcct gtatgattta ttggatgtt
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