英文名称： pCANTAB5E, pComb3噬菌体展示系统载体质粒图谱序列抗性说明书
试剂级别： 分子生物学级
pCANTAB5E, pComb3噬菌体展示系统载体质粒图谱序列抗性说明书.辅助噬菌体M13K07, VCSM13.宿主菌TG1，DH5aF,XL1-Blue
Product Information Order ID Name Description Biovector106283 pCANTAB 5E pCANTAB 5E,DNA. 15uL,AmpR.Storage:-20℃ Transformation of plasmid DNA to competent E. Coli cells 1. Thaw competent cells on ice. 20–200µL per tube 2. Add max. 1-3µL of plasmid 3. Mix very gently! 4. Incubate the tubes on ice for 30 min 5. Heat shock the cells for 45 sec to 2 min at 42°C 6. Place the tubes immediately on ice for at least 2 min 7. Add 800µL of SOC medium to each tube 8. Incubate for 1 hour at 37°C and shake vigorously 9. Spin down briefly and remove most supernatants 10. Resuspend cell pellet with the rest SOC medium in the tube by pipetting 11. Plate out the suspension on a LB agar plate containing the appropriate antibiotic. Incubate the plates overnight at 37°C 
Product Data Sheet
Order ID | Name | Description |
Biovector108925 | pComb3 | pComb3,AmpR. in E.coli. Storage:4℃ |
Recovery
1. Obtain an LB agar plate with the appropriate antibiotic.
2. Using a sterile pipette tip, touch the bacteria growing within the punctured area of the stab culture. (A sterilized wire loop or sterile toothpick can be used in place of a sterile pipette tip.)
3. Run this tip lightly over a section of the plate, as shown in the figure, to create streak #1.
4. Using another sterile pipette tip, pass through streak #1 and spread the bacteria over a second section of the plate, to create streak #2.
5. Using a third sterile pipette tip, pass through streak #2 and spread the bacteria over the last section of the plate, to create streak #3.
6. Grow overnight in a 37 o C incubator (unless a different growth temperature is indicated on the plasmid datasheet).
7. In the morning, single colonies should be visible. If the bacterial growth is too dense, re-streak onto a new agar plate to obtain single colonies.
Product Information
Order ID | Name | Description |
Biovector-01128569 | M13KO7 | M13KO7 helper phage, 100uL. Storage:-20℃ |
Description:
M13KO7 is an M13 derivative which carries the mutation Met40Ile in gII, with the origin of replication from P15A and the kanamycin resistance gene from Tn903 both inserted within the M13 origin of replication (1). M13KO7 is able to replicate in the absence of phagemid DNA. In the presence of a phagemid bearing a wild-type M13 or f1 origin, single-stranded phagemid is packaged preferentially and secreted into the culture medium.This allows easy production of single-stranded phagemid DNA for mutagenesis or sequencing.
Source:
M13KO7 phage supernatant was isolated from infected E. coli ER2738 by a standard procedure.
Concentration:
1 x1011 pfu/ml
Storage Temperature:
-20°C
Use of M13KO7 Helper Phage for isolation of single-stranded phagemid DNA
Protocol
1. Transform phagemid vector into appropriate F strain (CJ236 for Kunkel mutagenesis).
2. Inoculate 50 ml LB (no antibiotic) with a fresh colony, grow at 37°C with vigorous aeration until slightly turbid (<10 Klett, A600 < 0.05). For Kunkel mutagenesis, add uridine to 0.25 µg/ml.
3. Add 50 µl M13KO7 helper phage (final concentration of 1 x 108 pfu/ml), continue vigorous aeration for 60-90 minutes.
4. Add kanamycin to final concentration of 70 µg/ml, grow overnight (14-18 hours) with vigorous aeration.
5. Spin culture at 8,000 rpm for 10 minutes. Transfer supernatant to a new tube and spin again.
6. Pipet the upper 90% of supernatant into a new tube. To this supernatant, add a 0.2 volume of 2.5 M NaCl/20% PEG. Incubate at 4°C for 60 minutes.
7. Recover the phage by centrifugation at 8,000 rpm for 10 minutes. Decant supernatant and spin again briefly.
8. Resuspend the pellet in 1.6 ml TE, transfer to 2 microfuge tubes.
9. Spin in a microfuge for 1 minute to pellet any remaining cells, transfer supernatant to new tubes.
10. Add 200 µl of the 2.5 M NaCl/20% PEG solution to each, let sit at room temperature for 5 minutes, spin in a microfuge for 10 minutes.
11. Decant the supernatant, spin again briefly, remove last traces of supernatant with pipetman.
12. Resuspend each pellet in 300 µl TE. Extract with phenol (let sit 15 minutes before spinning), then phenol/chloroform (50/50: v/v; twice), and finally chloroform. Add 30 µl 2.5 M NaOAc, pH 4.8 and alcohol precipitate.
13. Suspend the dried pellets in 25-50 µl TE. Yield should be >50 µg single-stranded phagemid for pUC origin vectors.
Amplification of M13K07 helper phage.
1. Inoculate a single colony of E. coli strain XL1-Blue (MRF) into 10 ml LB broth containing 12.5 mg/ml tetracycline.
2. Incubate at 37℃/225 rpm until OD600 = 0.3 (equivalent of approx. 1.6 x 108 cfu/ml).
3. Add M13K07 helper phage at a ratio of 1:20, bacterial cells to phage.
4. Incubate at 37℃ for 30 min without shaking.
5. Add kanamycin to a final conc. Of 25 mg/ml and incubate at 37℃/225 rpm for 8 hours.
6. Incubate at 65℃ for 15 min and centrifuge at 4000 rpm for 15 min.
7. Aliquot the supernatant, titre and store at -20℃.
Order ID | Name | Description |
BV01128569 | VCSM13 | Helper Phage VCSM13, 100uL.Store -20°C |
Description
Interference-resistant helper phage (6 kb, single stranded), Derived from M13 K07 mutant, Carries kanamycin-resistance gene in IG region, High rescue efficiencies with kanamycin selection
Phage Amplification
If the titer drops over time, grow up XL1-Blue cells in 10 ml of media to an OD600 of 0.3 and add VCSM13 helper phage at a multiplicity of infection of 20:1 (phage-to-cell-ratio). Grow at 37°C for 30 minutes and then add kanamycin to a final concentration of 70 µg/ml. Continue growing at 37°C for 8 hours, heat to 65°C for 15 minutes and spin down the cell debris.
Titer of the supernatant should be between 1 × 1011 and 1 × 1012 pfu/ml. For further details about phage amplification or titering, please see Reference 1.
Reference 1.
Sambrook, J., Fritsch, E. F., and Maniatis, T., eds. (1989) “Molecular Cloning: A Laboratory Manual,” Second Edition, 4.21-4.23.
Product Information
Order ID | Name | Description |
Biovector-01128569 | XL1-Blue TG1 | XL1-Blue,200uL,TetR. Storage:4℃ TG1,200uL,TetR. Storage:4℃ |
Genotype:
XL1-Blue:
recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F´ proAB
lacIqZΔM15 Tn10 (Tetr)]
TG1:
F’[traD36 lacIq ∆(lacZ) M15 proA+B+] glnV (supE) thi-1 ∆(mcrB-hsdSM)5 (rK- mK- McrB-) thi ∆(lac-proAB)
Description:
The XL1-Blue strain allows blue-white color screening for recombinant plasmids and is an excellent host strain for routine cloning applications using plasmid or lambda vectors.
XL-1 cells are tetracycline resistant. XL1-Blue cells are endonuclease (endA) deficient, which greatly improves the quality of miniprep DNA, and are recombination (recA) deficient, improving insert stability. The hsdR mutation prevents the cleavage of cloned DNA by the EcoK endonuclease system. The lacIqZΔM15 gene on the F´ episome allows blue-white color screening.
TG1 strain is particularly useful for phage display protein expression. TG1 cells are also suitable for M13 phage work, general cloning, blue/white screening and protein expression.
Recovery
1. Obtain an LB agar plate with the appropriate antibiotic.
2. Using a sterile pipette tip, touch the bacteria growing within the punctured area of the stab culture. (A sterilized wire loop or sterile toothpick can be used in place of a sterile pipette tip.)
3. Run this tip lightly over a section of the plate, as shown in the figure, to create streak #1.
4. Using another sterile pipette tip, pass through streak #1 and spread the bacteria over a second section of the plate, to create streak #2.
5. Using a third sterile pipette tip, pass through streak #2 and spread the bacteria over the last section of the plate, to create streak #3.
6. Grow overnight in a 37 o C incubator (unless a different growth temperature is indicated on the plasmid datasheet).
7. In the morning, single colonies should be visible. If the bacterial growth is too dense, re-streak onto a new agar plate to obtain single colonies.