Although there is theoretically no limit to the number of fragments of exogenous DNA that can be carried by a recombinant M13 phage, in practice there is a limit: long fragments of exogenous DNA are more susceptible to deletions and rearrangements than short fragments. Therefore, if possible, it is preferable to clone no more than 1000 bases of DNA fragments into the M13 phage. Also, when sequencing DNA with "forward" or "reverse" universal sequencing primers, the center region of a large fragment may lie outside the region that can be measured. This experiment was derived from "Molecular Cloning Laboratory Guide, Third Edition", translated by Huang Peitang et al.
Operation method
Cloning of M13 phage vector
Principle
Although there is theoretically no limit to the number of fragments of exogenous DNA that can be carried by a recombinant M13 phage, in practice there is a limit: long fragments of exogenous DNA are more susceptible to deletions and rearrangements than short fragments. Therefore, if possible, it is preferable to clone no more than 1000 bases of DNA fragments into the M13 phage. Also, when sequencing DNA with "forward" or "reverse" universal sequencing primers, the central region of a large fragment may lie outside the region that can be measured.
Materials and Instruments
Phage T4 DNA ligase Restriction endonuclease Exogenous DNA Test DNA M13 Phage vector DNA Receptor cells of appropriate E. coli strains with F' plasmid E. coli F' pavement bacteriophage Move I. Materials For more product details, please visit Aladdin Scientific website.
ATP Ethanol IPTG Phenol Chloroform Sodium acetate TE X-gal
Agarose gel YT or LB agar plate YT or LB medium YT or LB upper agar or agarose Culture tube Heater Ice bath Water bath
1. Buffers and solutions
ATP (10 mmol/L)
Ethanol
IPTG ( 20%, m/V)
Phenol/chloroform (1:1, V/V)
Sodium acetate (3 mol/L, pH 5.2)
TE (pH 7.6 and pH 8.0)
X-gal (2%, m/V)
2. enzymes and buffers
Phage T4 DNA Ligase
restriction endonuclease
3. gels
Agarose gel (0.8%) suspended in 1 X TBE containing 0.5 μg/ml ethidium bromide.
4. nucleic acids and oligonucleotides
Exogenous DNA
Test DNA
5. Culture media
YT or LB agar plates
YT or LB medium
YT or LB top agar or agarose
6. Specialized equipment
Culture tubes (5 ml or 15 ml, e.g. Falcon 2054 or 2006, Becton Dickinson), pre-cooled to 0°C
Heater, set to 47°C
Ice bath
Water bath, set to 12~16°C and 42°C
7. Carriers and strains
M13 phage vector DNA (RF)
Receptor cells of appropriate E. coli strains with F' plasmid
E. coli F' planktonic organisms
II. Methods
Preparation of vector DNA
1. Completely digest 1~2 μg of M13 phage RF DNA with 3~5 times excess of appropriate restriction enzymes, and set up a control containing M13 RF DNA without restriction enzymes.
2. At the end of the incubation, take a small sample of DNA (50 ng) from each tube and analyze the digestion by 0.8% agarose gel electrophoresis. If digestion is incomplete (e.g., there is still visible closed loop DNA), add more restriction enzyme and continue incubation.
M13 RF DNA products may contain some amount of single-stranded M13 DNA, which appears as fast-migrating fuzzy bands on agarose gel electrophoresis. Since single-stranded DNA is not cleaved by most restriction enzymes, these bands should be consistent between control and sample.
3. After complete digestion, the M13 DNA is extracted with phenol/chloroform and purified by standard ethanol precipitation methods in the presence of 0.3 mol/L sodium acetate (pH 5.2), and the DNA is dissolved in TE (pH 8.0) at a concentration of 50 μg/ml.
4. If desired, dephosphorylate the linear vector DNA by treatment with calf alkaline phosphatase or shrimp alkaline phosphatase. At the end of the dephosphorylation reaction, the alkaline phosphatase is inactivated by heat or digestion with proteinase K, followed by phenol:chloroform extraction.
5. Recover linear M13 DNA by the method of step 3. The dephosphorylated DNA is dissolved in TE (pH 7.6) at a concentration of 50 μg/ml.
Preparation of exogenous DNA for cloning
6. Independent exogenous DNA restriction fragments can be obtained by appropriate restriction enzyme digestion and purification by agarose gel electrophoresis. The prepared exogenous DNA is dissolved in TE (pH 7.6) at a concentration of 50 μg/ml.
Ligation
To ligate complementary sticky ends, follow steps 7 to 9 below to set up a flat-end ligation reaction, see the protocol "Cloning of Flat-Ended Fragments in Plasmid Vectors".
7. In a microcentrifuge tube (Tube A), mix approximately 50 ng of vector DNA with a molar 1-5 times excess of target (exogenous) DNA in a volume of no more than 8 μl. If necessary, adjust the volume to 7.5-8.0 μl by adding TE (pH 7.6): 
8. Add 1 μl of 10X Ligation Buffer and 1 μl of 10 mmol/L ATP to each of all four reaction tubes (tubes A~D).
9. Add 0.5 Weiss unit of phage T4 DNA ligase to each of tubes A, B and D. Tap the wall of each tube for a few seconds. Tap the wall of each tube for a few seconds to mix the contents. 12~16℃ for 4~16 hours.
Transformation
10. Use YT or LB medium at 37℃ with continuous shaking to prepare the plates for overnight culture.
11. Take one sensory cell of the desired strain with F' plasmid from -70℃ refrigerator, melt it at room temperature and put it on ice for 10 min.
12. Add 50~100 μl of F' bacterial sensory cells to each of 16 sterile 5 ml culture tubes (Falcon 2054, Becton Dickinson) pre-cooled to 0℃.
13. Immediately add 0.1, 1.0 and 5 μl each of ligation product and control (tubes A~D) to each tube containing receptor cells. tap the wall of the tube for a few seconds to mix the bacteria and DNA. place the tube on ice for 30~40 min. set up two transformation controls, one with 5 pg of M13 phage RF DNA, and the other without DNA.
14. While the ligated DNA is incubating with the receptor cells, prepare 16 sterile culture tubes containing 3 ml of melted YT or LB upper agar and place on a heater or water bath at 47°C until step 16.
15. Transfer the culture tubes containing the susceptible bacteria and DNA to a 42°C water bath, incubate, and immediately transfer the tubes back to the ice water bath.
Transformed Cell Plate
16. Add 40 μl of 2% X-gal, 4 μl of 20% IPTG and 200 μl of overnight cultured E. coli cells (step 10) to the culture tubes prepared in step 14 containing melted top agar and mix the contents by gentle shaking for a few seconds. Add each transformed cell into each culture tube, cover with a lid and invert three times, pour the contents of the prepared tubes to the labeled LB agar plates in order, and rotate the plates so that the bacteria and the top layer of agar are spread evenly.
It may be difficult to get the top layer of agar to spread completely on the agar plate, especially if you are using a large plate (15 cm diameter) for the first time. Pre-warming the agar plate at 37°C for 30-60 min before spreading can slow down the solidification of the upper agar layer (and thus allow more time for turning the plate). Upper agar temperatures above 47°C will kill sensory bacteria (and significantly reduce transformation rates).
17. Cover the plate and allow to solidify at room temperature for 5 min, blot condensation from the plate cover with a Kimwipe tissue, invert the plate, and incubate at 37℃. 