Establishment of randomized overlapping DNA insertion libraries
Establishment of randomized overlapping DNA insertion libraries
The following protocol describes the starting steps of birdshot sequencing, from shearing of target DNA to construction of a DNA fragment library with M13 phage vectors, and also includes the two methods of breaking the target DNA, sonication and spraying, as well as the technical points of repairing the ends of the fragments with DNA polymerase. This lab is from the next volume of the Laboratory Guide to Molecular Cloning (Third Edition) by [American] J. Sambrook D.W. Russell.
Operation method
Establishment of randomized overlapping DNA insertion libraries
Materials and Instruments
Ammonium acetate ATP Ethanol Glycerol Distilled water IPTG MgCl2 NaCl Phenol Polyethylene glycol TE TM buffer Tris-HCl TTE buffer X-gal T4 phage DNA ligase T4 phage DNA polymerase T4 phage polynucleotide kinase Escherichia coli DNA polymerase Agarose gel Polyacrylamide gel Phage DNA DNA Molecular quality standards dNTP solution Target DNA LB or YT medium Move Materials For more product details, please visit Aladdin Scientific website.
Centrifuges and rotors Deep-well microplates, lids, caps Microtitre plates Multi-tube pipettes Multi-tube vortex oscillating mixers Silver tape Ultrasonicator or sparger Toothpicks Water baths Escherichia coli receptor cells M13 phage vector DNA
buffers and solutions
Ammonium acetate (10 mol/L)
ATP
Ethanol
Glycerol (sterile, 100%)
Water (deionized distilled water)
IPTG
MgCl2
NaCl
Phenol (pH 7.6 Tris saturated)
Phenol: chloroform (1:1, m/V)
Polyethylene glycol (30% m/V PEG 8000) Dissolved in 2.5 mol/L NaCl
Optional, see step 1
Polyethylene glycol (20% m/V PEG 8000) in 2.5mol/LNaCl
TE (pH 7.6)
10XTM buffer
0.5mol/LTris-HCl(pH8.0)
150 mmol/LMgCl 2
TTE buffer
Contains 0.5% Triton X-100TE (pH 8.0)
X-gal
Enzyme and Buffer
T4 phage DNA ligase
T4 Phage DNA Polymerase
T4 phage polynucleotide kinase
E. coli DNA polymerase I Klenow fragment
Gel
Agarose gels (two 1% and one 0.7%)
Polyacrylamide gel (neutral, 5% polyacrylamide) or agarose gel (0.8% low melting agarose)
Nucleic acids and oligonucleotides
λ phage DNA or Φ174 DNA completely digested with Alu I [1ug Alu I, dissolved in 20ul TE (pH 7.6)].
After digestion, the DNA was extracted with an equal volume of phenol/chloroform and the restriction endonuclease was removed. The ethanol-precipitated DNA is redissolved in TE (PH7.6) and the efficiency of ligation of the digested DNA to the dephosphorylated, linearized construct is tested according to step 13. Digestion of the DNA is not required if using commercial implants.
DNA Molecular Quality Standards
Appropriate size DNA molecular quality standards, such as PUC18pUC19DNA digested by Life Technologies with Sau3AI, or a gradient molecular quality standard with a difference of 123bp; ΦX174 DNA digested by New England Biolabs with Hae III.
dNTP solution containing four dNTPs, each at a concentration of 2.0 mmol/L.
Target DNA
Target DNA preparation is usually obtained by digesting recombinants constructed from high-capacity implants (e.g., mucoid implants, BAC, PAC, or P1) with restriction enzymes. The cleavage site of the restriction enzyme used should not be present in the sequence of the cloned fragment. If possible, try to use a restriction enzyme that produces sticky ends, which simplifies the ligation of purified poop DNA (step 1). After the target DNA fragments have been enzymatically cut from the vector, purify them by electrophoresis on a low-melting-point agarose gel (see Scheme 6 or 7 in Chapter 5). The purified DNA is dissolved in TE (pH 7.6) at a concentration of 50 ng for the DNA solution, and the integrity and recovery of the DNA is checked by agarose gel electrophoresis.
Medium
LB or medium containing 5 mmol/L MgCl 2 2xYT medium with 5 mmol/L MgCl2
Top layer of agarose
YT agar plate
Centrifuges and rotors
Centrifuge for centrifuging deep well microplates and standard microtitre plates at 3000r/min.
Examples include Beckman GPR centrifuges with microplate racks and Jocan CR422 centrifuges with pendulum heads (type M4) and microplate racks.
Specialized equipment
Deep-well microplates, lids and caps
Microtiter wells should be able to hold 1~1.2 ml, and withstand centrifugation at 3000r/min. Such as 96-well flat-bottom plates (DBM Scientific, San Fernando, California) and caps (V3-Verl-Tips-Cover, Ulater Sciemifici purchased from Baxter Inc., McGaw Park, Illinois). Beckman 96-tube cassettes with monolithic lids and caps were also available. Preparation of 96 M13 phage templates requires two cassettes of tubes with caps and caps.
Microtitre plates (96 wells with appropriate caps)
See the section on Microtitre plates in the Information section.
Multi-tip pipettes (8 or 12 tips)
Multi-tube vortex oscillating mixers
Silver tape (3M product) or equivalent items
The tape must ensure that the 96-tube box is sealed without leakage. Silver tape from 3M is available in the U.S. and Mexico from R.S. Hughes (http://www.rshughes.com).
Ultrasonograph or clearing spray
With regard to ultrasound, either a micro-probe sonicator or a cup-angle type sonicator can be used. A cup-angle sonicator (e.g., XL2015 sonicator equipped with the CL4 cup-angle probe, a Heat Systems product) is recommended for this protocol for two reasons: 1. DNA is placed in microcentrifuge tubes, which are usually only 20--25ul in volume; and 2. there is no contamination of the cup-angle cup sonicator probe because it does not come into contact with the DNA solution. With regard to nebulizers, a method for adapting disposable therapeutic nebulizers can be found in Appendix 8.
Toothpicks.
Adjustable water baths of 16°C, 37°C, 68°C, and 80°C.
Carriers and strains
M13 phage vector DNA, including blue-white screened, linearized, flat-ended and dephosphorylated vector DNA.
These types of preformed cultivar DNA can be purchased from companies or prepared according to additional methods provided later in this program.
E. coli receptor cells of appropriate strains (e.g., XLIF'-Blue, DH5aF')
Highly efficient E. coli receptor cells (>109 transfectants/ug closed-loop planted DNA) are important for obtaining high abundance of target DNA libraries. Pre-prepared high-efficiency E. coli receptor cells can be purchased from the company or can be prepared by referring to Methods 23~26 in Chapter 1. If an electrotransfer apparatus is used, we recommend the use of electrotransferred E. coli receptor cells for efficient transformation.
Methods
Target DNA self-ligation
Self-ligation is required to ensure that the end sequence of the raked DNA is present in sufficient amounts in the fragments used to construct the library.
1. Add 5-10 g of purified target DMA to a clean centrifuge tube with.
10XT4 Phage DNA Ligase Buffer 2.5ul
5 mmol/LATP 2.5ul
30% m/V PEG8000 (optional) 5.0ul
T4 Phage DNA Ligase 0.5~2.0Weiss Unit
H20 to final volume 25ul
The mixture is incubated at 16°C for 4 h, then heated at 68°C for 15 min to inactivate the ligase.
If the target DNA is flat-ended, the addition of PEG to the reaction can improve the ligation efficiency. For sticky-end ligation, the amount of ligase used is 0.5Weiss unit; for flat-end ligation, the amount of ligase used is 2.0Weiss unit.
Some commercial ligase buffers contain ATP, so it is not necessary to add ATP.
2. Add 175ul TE (pH7.6), purify the ligated DNA by extraction with phenol: chloroform, precipitate the DNA with 2mol/L ammonium acetate and 3 times the volume of ethanol, and recover the DNA by centrifugation in a microcentrifuge for 5 min at the maximum speed, and then wash the precipitate with 0.5 ml of 70% ethanol at room temperature, and centrifuge again.
3. Remove as much of the supernatant as possible and allow the last traces of ethanol to evaporate at room temperature. Dissolve the DNA in a microcentrifuge tube with 25ul of TE (pH 7.6).
Breakage of target DNA
The following method is a modification of the Birren et al. (1997) method.
4. Sonication or spray treatment is used to break the target DNA into 0.8 to 1.5 kb long fragments (see Table 12-1).
Sonication breaks DNA (see Figure 12-3). 
a. Fill the corner cup of the sonicator with ice water and turn the sonicator on. Set the time and power to 10, and preheat the sonicator with two 40-second pulses.
Ice water prevents DNA denaturation and it is a good idea to change the ice water in the corner cup before sonicating different samples.
b. Place the centrifuge tube containing the DNA in ice water with the bottom of the tube 1 to 2 mm above the center hole of the probe in the corner cup and sonicate the DNA (see Figure 12-3).
Sonicate the test sample DNA and analyze the sonication results using the method described in step 4 to determine the optimal sonication conditions. For most DNA samples, the power is set to 3, and two 6-second pulses produce fragments of 500-2000bp size.
c. Rapidly centrifuge the sonicated DNA solution to the bottom of the centrifuge tube and place on ice.
d. Take lul of the sonicated DNA sample and the appropriate size DNA molecular mass standard and analyze the DNA fragment size by 0.7% agarose gel electrophoresis. The remaining DNA sample remains on ice during electrophoresis.
If the resulting DNA fragments are not of the appropriate size, change the sonication conditions and sonicate again. If the resulting DNA fragments are the right size, continue with DMA end repair as described in step 5.
Spraying Broken DNA (see Figure 12-4) 
a. Prepare the following DNA solution and place it in a spray cup.
DNA sample (from Step 3 ) 5 to 10ug
10XTM buffer (pH 8.0) 200ul
Sterile 100% glycerol 1 ml
Sterile water to a final volume of 2 ml
b. Place the DNA samples in an ice bath and spray the DNA samples under optimal conditions based on experience.
The size of the DNA fragments is largely determined by the pressure of the nitrogen. For example, 8 psi (0.56 kg/cm2) pressure treatment of mucoid DNA mainly produces fragments of 1000 to 2500 bp in size. The optimal spray pressure and spray time for a new target DNA is determined empirically. As with sonication, an ice water bath prevents DNA degradation and is important for producing uniformly sized DNA fragments.
C. Place the nebulizer in a suitable centrifuge rotor lined with polystyrene foam. Rapid centrifugation at 4°C at 2000 g (1000r/min in a centrifuge equipped with a microplate holder) deposits the sample DNA solution at the bottom of the nebulizer cup.
d. Divide the DNA sample solution into four portions and transfer to a 1.5 ml centrifuge tube. Precipitate the DNA with ethanol according to the standard method and vacuum dry the DNA precipitate.
e. Dissolve each portion of DNA precipitate with 35ul of TE (pH 7.6). Take 1ul of the spray-treated DNA sample and the appropriate molecular mass of DNA molecular mass standard, and analyze the size of DNA fragments by 0.7% agarose gel electrophoresis. The remaining DNA samples from electrophoresis are stored at 4°C.
If the DMA fragments obtained are not of the appropriate size, change the spray conditions and spray again. If the resulting DNA fragments are the right size, continue with DNA end repair as described in step 5.
DNA Repair, Phosphorylation and Size Selection
The DMA ends produced by spraying and sonication are highly heterogeneous, including flat and mutilated ends, and ends with and without phosphate groups. Because only a fraction of these molecules can be repaired by DNA polymerase, the efficiency of hydrostatically sheared DNA cloning to M13 phages is generally low. However, sonication of 5-10ug of target DNA, repair, and selection of appropriately sized fragments generally results in the formation of several thousand recombinant clones.
5. To the interrupted DNA (~25ul), add:
10XT4 Phage DNA Polymerase Buffer 4.0ul
2.0 mmol/L dNTP solution with four dNTPs 4.0ul
T4 Phage FJNA Polymerase 5 Units
Water to final volume 40ul
Incubate at room temperature for 15 min, then add approximately 5 units of Klenow Fragment and continue to incubate at room temperature for 15 min.
This reaction uses two DNA polymerases to repair damaged ends of DNA fragments resulting from hydrostatic shearing. t4 phage DNA polymerase flattens the 3' concave end and its exonuclease activity removes the protruding end. klenow fragments provide a second tool for flattening the 3' concave end. See the "E. coli DNA Polymerase I Klenow Fragment" section in the Information section.
6. purify the DNA by phenol/chloroform extraction. transfer the upper aqueous phase to a clean tube, bring the NaCl concentration to 0.1mol/L, add 2x the volume of ethanol, and recover the precipitated DNA. wash the DNA precipitate with 70% ethanol.
7. Add 25ul of TE (pH 7.6) to re-dissolve the precipitated DNA.
8. Mix the following components in a microcentrifuge tube:
Interrupted DNA 23ul
10x Polynucleotide Kinase Buffer 3ul
20 mmol/L.ATP 3ul
T4 Phage polynucleotide kinase 1 unit
T4 Phage Polynucleotide Kinase catalyzes the phosphorylation of the 5' end of the flat-end DNA, this step is not necessary. This step is not necessary, but generally improves the efficiency of ligation of the DNA fragments to the vector.
9. Incubate at 37°C for 30 min.
10. Purify DNA fragments of the desired size (0.8-1.5 kb) by 0.8% low melting point agarose gel electrophoresis or 5% neutral polyacrylamide gel electrophoresis (see Chapter 5).
To minimize the possibility of contamination, several lanes should be left empty between the broken target DNA and the standard reference, which is especially important when using flat-end DNA as a reference, as it ligates significantly more efficiently to vector DNA than to sheared target DNA.
11. Recover the target DNA from the gel using the method described in Chapter 5. Dissolve the purified DNA in 25ul of TE (pH 7.6).
12. Take 1ul of DNA and analyze the integrity and recovery of the purified DNA by 1% agarose gel electrophoresis.
Ligation to vector DNA
13. Set up a series of test ligation reactions containing 50ng (~0.01pmol) of linearized and dephosphorylated vector DNA.
DNA and increasing concentrations of flat, phosphorylated target DNA fragments (see Table 12-2). 
14. ligating solution by electrotransfer or transformation of E. coli receptor cells of the appropriate strain (see Methods 23-26 in Chapter 1). Spread the bacteria on medium containing IPTG and X-gal and incubate overnight at 37°C.
The purpose of this step is to find out the concentration of a target DNA fragment in order to minimize recombinants containing artificially fused target fragments, which would complicate sequencing. Therefore, when determining a large number of ligation reactions (step 16), care must be taken to avoid using saturating amounts of target DNA; instead, the amount of target DNA determined should be such that the number of recombinant clones is moderately elevated (approximately up to 5-fold) compared to the backmost.
15. Count the number of blue and white phage spots the following day.
The number of recombinants obtained using broken flat-end target DNA is approximately one-tenth of the number obtained using flat-end DNA prepared using restriction endonuclease {e.g., compared to λDNA digested with Alu I or ΦX174DNA as the starting ligation reaction).
16. Set up a larger-scale ligation reaction with a minimal amount of broken flat-end target DNA in order to obtain enough recombinant clones to complete the sequencing task. Transform E. coli with ligated DNA and incubate at 37°C overnight.
The purpose of this step is to ensure that at least five recombinant clones are obtained for each target fragment. Figure 12-5 shows the approximate number of clones that must be sequenced to cover 95% of the double-stranded target DNA sequence.
17, Collect the plates the following day and store the transformants under appropriate conditions. Methods for preparing DNA templates from a series of individual colorless phage spots are described in Method 4 of Chapter 3.
M13 recombinant phage phage spots should be picked and amplified as soon as possible (see the "Pick of Phage Spots" column of Scheme 2 in Chapter 3). Because phage particles can spread over long distances in the top layer of agar, phage spots that have been grown for long periods of time (>12-16 hours) at 37°C or that have been stored at 4°C for several days are often already contaminated. Therefore, the intensity and number of background bands increase when using DNA prepared from older phage spots for sequencing reactions.
Cultivation of M13 Phage Recombinant Clones in 96-Tube Cassettes
Single colorless phage spots were picked as described in Scheme 2 in Chapter 3 and inoculated in 15 ml tubes containing 2 ml of bacterial medium for incubation. The recombinant virus particles are then recovered separately and the DNA purified (see schemes 3 and 4 in chapter 3). Since only 12 or 24 clones can be processed at a time, the template purification process is time-consuming and tedious. Large-scale DNA sequencing requires thousands of DNA templates, but single-stranded DNA templates cannot be prepared quickly and economically using organic solvent extraction and multi-step centrifugation. Alternative methods for bulk preparation of M13 phage templates typically include purification of phage particles or a combination of automated technical equipment (e.g., please see Mardis and Roe 1989; Smith et al. 1990), filtration (Eperon 1996), magnetic beads (Alderton et al. 1992; Wahlberg et al. 1992; Wahlberg et al. 1992; Wahlberg et al. 1992; Wahlberg et al. 1992; Wahlberg et al. 1992; Wahlberg et al. 1992; Wahlberg et al. 1992; Wahlberg et al. 1992). Single-stranded DNA is purified by filtration (Eperon1996), magnetic beads (Alderton et al.1992; Wahlberg et al.1992; Hawkins et al.1994), or paramagnetic spheres (Fry et al.1992; Wilson1993), but the equipment and staff required to operate the equipment are not available in the average academic institution. However, Zollo and Chen (1994) reported an efficient and reproducible method for culturing M13 phage clones in 96-well plates and preparing single-stranded DNA, which satisfies the need for large amounts of template for automated fluorescence-based DNA sequencers. 
In birdshot sequencing, it is very efficient to use disposable flat-bottomed well plates or disposable tube kits to culture M13 phage recombinants in M less bacterial cultures and process 96 samples simultaneously. All subsequent steps related to single-stranded DNA template preparation can be performed in the same plate, reducing the effort of transferring clones from one step to the next and allowing a researcher to prepare 960 or more DNA templates in a single day (Pleasesee Zollo and Chen1994).
18. For each of the 96 clones, a single colony of an E. coli F' strain (e.g., XLl-Blue, XLl-Blue MRF', or HD5αF') was inoculated, one by one, into 100 ml of LLB or 2 x YT medium and added to a 500 ml volume culture flask. Incubate at 37°C for 6-8 h with 300r/min oscillation.
19. Add MgS04 to the culture broth to a final concentration of 5 mmol/L.
To maintain equilibrium and symmetry during centrifugation, the number of clones of M13 phage cultured is an even multiple of 96.
Mg2+ increased the yield of M13 phage and reduced the difference in growth rate between clones.
Each group of 96 M13 phage clones requires approximately 80 ml of cultured cells.
20. Transfer 0.8 ml of cell culture solution to each tube of the 96-tube cassette using a multichannel pipette.
21. Using gloves, inoculate each tube of the 96-tube cassette with a single colorless M13 phage with a sterile toothpick. Puncture the center of the phage spot with the toothpick and drop into the culture tube.
22. To avoid confusion, leave the toothpick in the tube until all 96 tubes have been inoculated.
23. When the last phage spot has been picked, remove all the toothpicks and close the culture tube box. Label the culture box and place it in a shaker at 37°C with 250-300r/min for 8-12 h. Repeat steps 20 and 21 if necessary.
If the incubation time is longer than 12 h, the prepared single-stranded template will be contaminated with a large amount of M13 phage double-stranded replicative DNA and/or chromosomal DNA. DNA derived from bacterial lysis will increase the chance of primer mismatches in the double deoxy sequencing reaction. Prolonged incubation will also increase the chance of DNA clone deletions and rearrangements. Therefore, proper incubation for a short period of time is critical to the success of this method.
Purification of M13DNA
24. Remove the culture cassette from the incubator and centrifuge for 20 min at 2400 g (3000-3250 r/min in a centrifuge with a microplate sleeve) to precipitate the organisms.
The primary preservation method for M13 phage clones is to add 25ul of 80% glycerol to 50ul of suspension, blow the mixed solution up and down with a measuring spacer, and store the plates at -80°C. These plates serve as a source of phage for infection if additional template DNA preparation is required. The last thing to emphasize in this protocol is the safe preservation of phage. there is a greater possibility of cross-contamination between tubes in this protocol, which has little effect on DNA sequencing, but is unacceptable for the preservation of seed stock.
25. Add 120ul of 2.5 ml/L NaCl solution containing 20% PEG8000 to each tube of a new 96-tube cassette using a multichannel pipette.
26. Carefully remove the centrifuge tubes from the centrifuge and add 0.6 ml of supernatant from each tube to the PEG/NaCl solution using a multichannel pipette.
IMPORTANT: It is important not to stir the bacterial precipitate during this step, as mixing of the bacteria will significantly reduce the quality of the DNA sequencing.
27. Cover the tubes containing the phage suspension and FEG/NaCl solution with 96-tube caps to ensure a strong liquid seal is formed, and invert the tubes several times to mix the contents. Leave the cassette at room temperature for 30 min, followed by an ice bath for 30-60 min.
Centrifuge the tubes at 28.2400 g (3000-3250 r/min in a centrifuge with a microplate sleeve) for 30 min and collect the precipitated phage. Remove all rows of test tubes and place them upside down in a sink to air-dry. A small amount of white precipitate was visible at the bottom of each test tube. Put the tubes back into the tube box.
29. When all tubes have been emptied, invert the cassette onto blotting paper for a few minutes to empty any remaining traces of liquid. Replace the blotting paper with fresh paper and place the inverted cassette and paper in a centrifuge at 300r/min for 3-5 min to remove the last traces of PEG/NaCl solution.
30. Remove the cassettes from the centrifuge and check the bottom of the tubes for phage deposits. Add 20ul of TTE buffer to each tube.
If the phage grows well, the precipitate is white and opaque; if it does not grow well, the precipitate is blue and opaque. If the precipitate is a brownish mass, it is probably due to the excessive amount of organisms taken in step 26. Sequencing of templates prepared from brown precipitates gives poor results.
31. Seal the mouth of the test tube with 3M silver tape and vigorously shake the cassette in a multitube vortex shaker for 15-30 min.
32. Rapidly centrifuge the cassettes so that the liquid settles to the bottom of the tube, remove the base of each 96-well cassette, and place the tubes in a water bath at 80°C for 10 min.
This step is to lyse the M13 phage pellet. Removing the base of the cassette ensures that all tubes are held at 80°C for 10 min.
33. Remove the tubes from the water bath and cool to room temperature. Reinstall the cassette base and rapidly centrifuge the cassette to allow the liquid to settle to the bottom of the tube.
34. Using a multichannel pipette, add 70ul of sterile water to the wells of the 96-well plate, transfer the phage lysate obtained in step 33 to the 96-well plate, and mix the two solutions by pipetting. Seal the 96-well plate with a piece of 3M silver tape, or cover it with a 96-well plate cover plate if sequencing is to be performed within 24-48 h. Label the plates. Label the plates and store at -20°C.
For each incubation, 2.5-5ul of M13 phage single-stranded DNA can be extracted.
35. Randomly take a DNA sample (5ul) from some wells and check the amount of DNA by 1% agarose gel electrophoresis.
If everything is normal, there is very little difference in DNA production from tube to tube. 2ul to 7.5ul of DNA is usually required for a DNA cycle sequencing reaction.
The amount of DNA required for the DNA cycle sequencing reaction is generally 2ul~7.5ul (see protocol 6). 