Partial cleavage (pre-reaction) of eukaryotic DNA for genomic libraries
Partial cleavage (pre-reaction) of eukaryotic DNA for genomic libraries
Regardless of the base composition and sequence of high-molecular-quality DNA, it can be fragmented in a semi-random fashion by hydrodynamic shearing. However, DNA prepared in this way requires a series of enzymatic reactions (end repair, methylation, junction attachment, junction digestion) to protect the internal restriction sites and to generate some sticky ends. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Partial cleavage (pre-reaction) experiments for eukaryotic DNA in genomic libraries
Principle
Regardless of the base composition and sequence of high-molecular-quality DNA, it can be fragmented in a semi-random fashion by hydrodynamic shearing. However, DNA prepared in this way requires a series of enzymatic reactions (end repair, methylation, junction attachment, junction digestion) to protect internal restriction sites and to generate some sticky ends.
Materials and Instruments
Restriction endonuclease Genomic DNA λ Phage DNA Plasmid Oligomers Move I. Materials For more product details, please visit Aladdin Scientific website.
Sucrose Gel Sampling Buffer Tris-Cl
Agarose gel Capillary tube Dialysis bag Water bath Wide mouth pipette tip
1. Buffers and solutions
Sucrose Gel Sampling Buffer
Tris-Cl (10 mmol/L, pH 8.0)
2. Enzyme and buffer
restriction endonuclease
3. gels
Agarose gel (0.6%) with 0.5X TBE containing 0.5 μg/ml ethidium bromide
Agarose gel for pulsed field gel electrophoresis
4. Nucleic acids and oligonucleotides
Genomic DNA, high molecular mass
λ Phage DNA and plasmid oligomers
5. Specialized equipment
Capillary tubes, sealing
Dialysis bags, boiled
preset in 70°C water bath
Wide mouth pipette tips
ii. Methods
1. Set up a pre-experiment using the same genomic DNA that will be used to prepare the cloned fragments.
(1) Dilute 30 μg of high molecular weight eukaryotic DNA to 900 μl with 10 mmol/L Tris-Cl (pH 8.0) and add 100 μl of appropriate 10X restriction buffer.
(2) Gently mix the solution with a sealed glass capillary tube to ensure that the high molecular mass DNA is completely and evenly distributed in the restriction enzyme buffer.
(3) After mixing, leave the diluted DNA at room temperature for 1 h to allow the residual DNA clusters to disintegrate completely.
2. Label microcentrifuge tubes with 1 to 10 markers. Transfer 60 μl of DNA solution into a microcentrifuge tube (tube 1) using a wide-mouth glass capillary or a disposable plastic pipette tip. Transfer 30 μl of DNA solution to the other 9 labeled microcentrifuge tubes. Place in an ice bath.
3. Add 2 units of appropriate restriction enzyme to tube 1.
4. Using a fresh pipette tip, transfer 30 μl of reaction solution from tube 1 to tube 2 of the centrifuge tube series. Mix as previously described and continue to the next tube. Leave tube 10 empty (no enzyme control) and discard 30 μl from tube 9.
5. Incubate at 37°C for 1 h.
6. Heat at 70°C for 15 min to inactivate the enzyme.
7. Cool the reaction to room temperature and add an appropriate amount of Sucrose Gel Sampling Buffer.
8. Using a wide-mouth plastic pipette tip or disposable glass capillary, transfer the solution to the spotting well of a 0.6% agarose gel, or better yet, to the loading tank of pulsed-field coagulation electrophoresis, followed by electrophoresis.
9. Compare the size of the digested eukaryotic DNA with DNA standards containing λ phage and plasmid oligomers. Identify some of the digestion conditions that yield large quantities of ideally sized genomic DNA. 