This protocol uses the nuclease BAL31 (purified from the marine bacterium Alteromonas espejiana BAL31) to produce unidirectional or bidirectional deletions in cloned DNA fragments.BAL31 is a complex enzyme that digests the double-stranded target DNA in an asynchronous manner.BAL31 therefore produces more heterogeneous deletions than processive enzymes such as exonuclease III (see Scheme 9). heterogeneity than processing enzymes such as exonuclease III (please see Scheme 9). This experiment is from the next volume of the Molecular Cloning Laboratory Guide (3rd edition) by J. Sambrook D.W. Russell.
Operation method
BAL31 Nuclease digestion assay for generating bidirectional deletion mutants experimental
Materials and Instruments
BAL31 Buffer EGTA Ethanol Phenol Chloroform Sodium Acetate Sucrose Gel Sampling Buffer TE Phage T4DNA Polymerase BAL31 Nuclease Escherichia coli DNA Polymerase IKlenow Fragment Restriction Endonuclease Agarose Gel Molecular Weight Standards for DNA for Gel Electrophoresis Move makings For more product details, please visit Aladdin Scientific website.
Meter Clock Water Bath
Buffers and solutions
Refer to Appendix 1 for the composition of storage solutions, buffers and reagents, and dilute the storage solution to the appropriate concentration.
5XBAL31 Buffer
2.5mol/L NaCl
62.5 mmol/LCaCl2
62.5 mmol/LMgCl2
100 mmol/LTris-Cl (pH 8.0)
There were 4 solutions of dNTP, each at a concentration of 0.5 mmol/L
EGTA (0.5 mol/L, pH 8.0)
ethanol
Phenol: chloroform (1:1, V/V)
Sodium acetate (3 mol/L, pH 5.2)
Sucrose Gel Sampling Buffer
TE (pH 7.6)
Enzyme and Buffer
Phage T4DNA Polymerase
BAL31 Nuclease
Escherichia coli DNA polymerase IKlenow fragment
restriction endonuclease
See steps 3, 23 and 31.
Gel
Agarose gel
See steps 3 and 32.
Agarose gel containing 0.5ug/ml ethidium bromide (0.8%)
See step 11.
Agarose Gel with 0.5ug/ml Ethidium Bromide prepared in TBE
See step 25.
Preparation of Agarose Gel
See step 27.
Nucleic acids and oligonucleotides
DNA molecular weight standards for gel electrophoresis
Specialty Equipment
Time Clocks
65°C water bath and water bath suitable for restriction endonuclease digestion temperature
Other reagents
The reagents required for Step 1 of this protocol are listed in Chapter 1, Options 17-19 or Chapter 3, Option 6.
The reagents required for Step 2 of this protocol are listed in Chapter 1, Option 9.
The reagents needed for Step 27 of this protocol are listed in Chapter 5, Options 4, 5, 6, or 7.
Step 30 of this protocol requires reagents listed in I.I. 1, Schemes 23~26 or Chapter 3, Schemes 6 or 8.
Step 31 of this protocol requires reagents listed in Chapter 1, Scheme 1 or Chapter 3, Scheme 3.
The reagents needed in step 34 of this protocol are listed in Chapter 12, Options 3, 4, and 5.
Methods
Preparation of Target DNA for BLA31 Digestion
1. Clone the target DNA fragments into a suitable plasmid or phage M13 vector.
If a deletion mutant is to be constructed from both ends of the target DNA, a suitable vector with multiple cloning sites should be selected to clone the target DNA into the vector in both directions.
2. purify the closed-loop re-tantalum DNA using a Qiagen chromatography column (or equivalent) and ethanol precipitation. re-dissolve the DNA in a minimum volume of Tris/EDTA solution.
It is important to use high purity closed-loop DNA to (i) reduce the proportion of contaminating RNA and small fragments of E. coli chromosomal DNA in the total end concentration of the reaction, and (ii) remove nicked loop DNA that is capable of degradation by BAL31.
3. completely digest 30ug of closed-loop DNA with restriction enzyme with the cleavage site at the end of the target DNA. define this site as the common site for the initiation of the nested deletion. Agarose gel electrophoresis was performed to verify complete restriction enzyme digestion.
4. purify the DNA by isocratic phenol/chloroform extraction. separate the organic and aqueous phases by centrifugation in a microcentrifuge at maximum speed for 3 min and transfer the aqueous phase to a new microcentrifuge tube.
5. Add 0.1x volume of 3mol/L sodium acetate (pH 5.2) and 2x volume of ethanol pre-cooled on ice. Allow to stand at O°C for 10 min, then centrifuge for 10 min at 4°C maximum speed in a microcentrifuge to harvest DNA.
6. Remove the supernatant and carefully rinse the DNA precipitate with 70% ethanol at room temperature. Dry the DNA precipitate at room temperature and solubilize the DNA precipitate to a lug/ul buffer with TE (pH 7.6). Store the DNA at -20°C.
BAL31 Activity Analysis
Most commercially available BAL31 enzyme products contain two kinetically distinct active forms of the enzyme: the fast form and the slow form (see the Information section on BAL31). The slow form is a protein hydrolytic degradation product of the fast form, and the rate at which BAL31 digests DNA is a function of the ratio of the fast form to the slow form in the particular enzyme preparation used. Purified fast enzyme preparations are available (Weietal.1983), but are expensive and often decay to the slow form during storage. In order to keep BAL31 in the fast form, the enzyme should not be frozen but stored at 4 °C.
Since the ratio of fast to slow forms in BAL31 varies from preparation to preparation, the enzyme activity of the particular batch used to construct the deletion must be determined as follows.
7. Mix in a microcentrifuge tube:
Linear DNA (1ug/ul) 4ul
Water 48ul
5XBAL31 buffer 13ul
Dispense the above mixture into 7 microcentrifuge tubes in the amounts indicated.
8. Make seven 2-fold dilutions of BAL31 with 1XBAL31 Buffer, preferably as follows: Dispense seven drops (2ul) of 1XBAL31 Buffer onto the surface of a paraffin membrane placed on a bed of ice or a cold metal block, and use a disposable micropipette tip to draw up 2ul of the BAL31 enzyme to be experimented on, mixing it with the first drop of BAL31 Buffer. With a new pipette tip, take 2ul of the above mixture of BAL31 and buffer and transfer it to the second drop of BAL31 buffer and mix it again. Do this until all of the BAL31 Buffer has been mixed with the enzyme. Quickly remove 1 to 6 microcentrifuge tubes containing linear DNA from each of the last 6 mixtures, leaving the 7th reaction tube without enzyme.
The concentration of most commercial BAL31 enzyme preparations is approximately 1 unit/ul; 0.05 to 0.1 units of BAL31 enzyme is sufficient to digest 1ug of 2kb long linear DNA molecules into fragments less than 200bp in length.
9. Incubate all microcentrifuge tubes (including those without enzyme) at 30°C for 30 min.
10. Add 1ul of 200 mmol/LEGTA (pH 8.0) to each tube and heat at 65°C for 5 min.
Ca2+ is required for the BAL31 reaction, and the enzyme activity can be completely inhibited by EGTA; heating at 65°C for 5 min also inactivates the enzyme.
11. Each of the above samples is mixed with 3ul of Agarose Gel Sampling Buffer and analyzed for DNA size by electrophoresis on an agarose gel containing 0.5ug/ml ethidium bromide (0.8%).
12. The gel is examined under UV light and the appropriate dilution of the enzyme is determined, i.e., just enough so that the DNA is digested moderately so that only small, diffuse fragments of DNA (200 bp) are detected. This dilution will be used in the large-scale digestion reaction (step 15).
Another way to check the extent of BAL31 digestion is to set up an SBAL31 mass digestion reaction and take samples at different time points. Each sample is digested with restriction endonuclease, which should be able to cleave the target fragment several times. As the BAL31 digestion progresses, the restriction fragments disappear in a certain order. The rate of BAL31 digestion can be estimated from the size and position of the restriction fragments. The amount of BAL31 used in a bulk digestion reaction should be sufficient to reduce the length of the target fragment by 20% within the first 5 min of the reaction.
Bulk Digestion with BAL31
13. Mixing
Linear DNA (1ug/ul) 20ul
Water 240ul
5XBAL31 buffer 65ul
Incubate the above mixture in a 30°C water bath.
14. During incubation at 30°C, prepare 8 microcentrifuge tubes, each containing 5ul of 200 mmol/LEGTA (pH 8.0), and label the tubes with time periods of 1.5 min, 3 min, 4.5 min, etc.
15. Add 36ul of diluted BAL31 (see Step 12) to the reaction mixture in Step 13, tap the walls of the centrifuge tube to mix the contents quickly and homogenize, then return to the 30°C water bath and start the timer.
16. Transfer 45ul of the reaction mixture to the appropriate labeled microcentrifuge tube every 1.5 min and place the tube on ice until all samples have been collected.
17. Heat at 65°C for 5 min to inactivate BAL31 enzyme.
18. Add 5ul of 3 ml/L sodium acetate (pH 5.2) to each tube, add 100ul of ice-cold ethanol, shake the mixture, and place the tubes on ice for 20-30 min.
19. Recover the DNA by centrifugation at 4°C maximum speed, remove the supernatant, wash the precipitate with 200ul of ice-cold 70% ethanol and centrifuge for 2 min.
20. Carefully remove the supernatant and leave the centrifuge tube with open cap upright at room temperature until all the ethanol has evaporated. Dissolve each portion of the precipitate in 23ulTE (pH 7.6).
Isolation of truncated target fragments
21. add to each portion of the DNA preparation:
0.5 mmol/LdNTP solution 3ul
10X polymerase buffer 3ul
Phage T4DNA polymerase (about 5 units) 1ul
React for 15 min at room temperature, then add 1 (~5 units) of Klenow fragment. Continue incubation at room temperature for 15 min.
The use of two different DNA polymerases in the repair reaction increases the recovery of mutants nearly threefold.
22. purify the DNA by phenol/chloroform extraction, and precipitate the DNA with ethanol as described in steps 18-20. dissolve each portion of DNA in 16ulTE (pH 7.6).
23. Add 2ul of the appropriate 10x Restriction Enzyme Buffer and 8 units of Restriction Enzyme to each portion of DNA to separate the target DNA from the vector and incubate the reaction for 1h at the appropriate temperature.
24. At the end of the incubation, remove 3ul from each digestion reaction and transfer to a new microcentrifuge tube, leaving the remaining digestion reaction on ice for use in step 27.
25. Mix 1ul of Sucrose Gel Sampling Buffer for every 3ul of the above reaction solution and add the sample to the agarose gel spiking wells containing 0.5ug/ml ethidium bromide and 0.5XTBE. An appropriately sized molecular weight standard reference should be added to the wells on the side of the gel.
The concentration of the filled agarose gel should be appropriate for the separation of target fragments and cultivars (see Table 5-2 in the Introduction section of Chapter 5).
26. Separate the target fragment from the vector DNA using gel electrophoresis, observe the gel under UV light, and determine which sample has been digested to the appropriate size by BAL31.
27. Combine plasmid samples containing target DNA of the appropriate size, and isolate and recover the target DNA fragments on preparative gels as described in Schemes 4-7 in Chapter 5.
28. Estimate the amount of target DNA purified based on the fluorescence intensity produced by ethidium bromide.
Cloning of missing target fragments
29. Ligate the missing poop fragment with a plasmid, phage, or phage M13 vector (see Chapters 1 or 3). The vector should carry a blunt end and an end matching the endonuclease used in step 23.
The exact composition and volume of the ligation reaction depends on the amount of target DNA, and if possible, 50ng or 100ng of target DNA should be used. ensure that the molar ratio of vector DNA to target DNA is at least 5 (in order to minimize the number of recombinants containing more than one fragment of target DNA). To maximize the formation of recombinants, the ligation reaction should be performed under conditions suitable for blunt-end ligation, e.g., in a small reaction volume with high concentrations of phage T4DNA ligase, polyethylene glycol, and low concentrations of ATP. For details on ligation conditions, see Scheme 19 in Chapter 1.
30. Transform (plasmid or phage) or transfect (phage M13 replicative DNA) suitable E. coli receptor strains with small amounts or dilutions of the ligation mixture. On the second day, randomly select 12 transformed colonies or phage M13 replicative DNA.
phage spots were randomly selected on the second day and cultured in small numbers.
31. Purify plasmid, phage, or phage M13 replicative DNA from the 12 cultures using one of the methods described in Chapters 1 or 3. Digest the DNA with a restriction enzyme that can cut the target fragment from the vector.
32. Analyze the size of the target fragment cut from each DNA by gel electrophoresis and a standard reference of appropriate size for DNA molecular weight.
33. If the results are satisfactory (e.g., the size of the target fragment is within the desired range), pick large numbers of transformed single colonies or phage spots that are independent of each other. Identify the size of the insert fragment as described above. Retain the recombinant subcultures carrying the size match.
34. Determine the exact site of the deletion site for each mutant by DNA sequencing (see Scheme 3, 4 or 5 in Chapter 12).