Ligation of plasmid and target DNA in low melting point agarose experiment
Ligation of plasmid and target DNA in low melting point agarose experiment
The most time-consuming step of the plasmid cloning operation is the purification of the desired sized fragments of exogenous and plasmid DNA by electrophoresis, which can be used to ligate the plasmid to the exogenous DNA in the presence of low-melting-point globular sugar.
Operation method
Experiments for ligating plasmids and target DNA in low melting point agarose
Principle
The most time-consuming step of the plasmid cloning operation is the purification by electrophoresis of the desired sized fragments of exogenous and plasmid DNA, and in the following scheme (taken from Struhl 1985), ligation of the plasmid and the exogenous DNA can be accomplished in the presence of low-melting-point globular lipids.
Materials and Instruments
Restriction endonuclease Exogenous DNA fragments Plasmid DNA Move I. MATERIALS Caveat The above mentioned ligation products can be used directly for transformation of E. coli or electroporation. Before transformation or electroporation, the microcentrifuge tube containing the ligation product should be heated at 70℃ for 10~15 min to re-melt the solidified gel. Common Problems Customarily, 1 to 5 μl of ligation product can be used to transform chemically prepared cells, while the more efficient electroporation method requires only 0.1 to 1.0 μl of ligation product. The use of larger volumes for electroporation will increase the concentration of solutes, making the onset of arcing difficult to predict. For more product details, please visit Aladdin Scientific website.
Tris-Cl MgCl2 DTT ATP T4 Phage DNA Ligase
Low melting point agarose gel Thermostat plate Portable long wave UV lamp Water bath
1. Enzymes and buffers
(1) 2X T4 phage DNA ligase reaction mixture:
1 mol/L Tris-Cl ( pH 7.6) 1.0 ul, 100 mmol/L MgCl2 2 .0 ul, 200 mmol/L DTT 1.0 ul, 10 mmol/L ATP 1.0 ul, H2O 4.5 ul, T4 phage DNA ligase 1.0 Weiss unit.
Each ligation reaction requires 10 ul of system.
The system should be prepared freshly before each application, pre-cooled on ice in microcentrifuge tubes, and the reaction mixture should be set aside on ice.
(2) Restriction endonuclease
2. Gel
Low melting point agarose gel
3. Nucleic acids and oligonucleotides
Exogenous DNA fragments, plasmid DNA (~100 ug/ml, dephosphorylated)
Approximately 100 ng of dephosphorylated DNA is required for each ligation reaction.
4. Specialized equipment
Thermostat plate that can be set to 70°C, handheld long-wave UV lamp (302 nm), and a water bath that can be set to 16°C. The method is as follows.
Methods
1. Set up an enzyme digestion system (no more than 20 ul), digest a certain amount of target DNA with appropriate restriction enzymes, and finally obtain 250 ng of target fragments.
2. Separate the DNA fragments by electrophoresis on a low melting-point agarose gel.
3. Examine the electrophoresis of the DNA in the gel under the long-wave UV lamp, and estimate the amount of DNA according to the fluorescence intensity of the bands, and cut off the target bands with a clean surgical blade, as little as possible. Use a clean scalpel blade to cut off the target bands, and cut as little agarose as possible (usually about 40~50 ul). Leave a small amount of each target band in the gel, which is used to indicate the position of each band in the gel electrophoresis in the photograph.
4. Load the cut pieces of gel into labeled microcentrifuge tubes.
The cut gel pieces can also be stored in sealed microcentrifuge tubes at 4°C for a few days.
5. Place the tube in a thermostat block at 70°C for 15 min to melt the gel strips. Determine the volume of gel in the centrifuge tube and calculate the volume needed to contain approximately 200 ng of DNA.
The aim is to collect 200 ng of DNA in a volume of 10 ul or less, depending on the situation. For bands below 10 (just visible in the gel), ligation can still be performed, albeit less efficiently.
6. Preheat a sterile microcentrifuge tube to 37°C and immediately add the following:
60 fmol of dephosphorylated plasmid DNA and 120-240 fmol of the exogenous DNA fragments (not to exceed 10 ul volume).
Mix the above mixture quickly with a sterile disposable pipette tip to avoid solidification of the agarose gel.
In the ligation reaction, the molar ratio of exogenous DNA fragments to plasmid vector is generally between 2:1 and 4:1.
7. For each reaction tube, make two controls accordingly, one with dephosphorylated plasmid vector only, and the other with exogenous DNA fragments only.
8. The reaction tubes are incubated at 37°C for 5-10 min, and then 10 ul of the now-cooled 2X T4 phage ligase reaction mixture is added to each tube. reaction mixture to each tube. Mix the mixture quickly with a sterile disposable pipette tip to avoid solidification of the agarose gel. incubate at 16℃ for 12-16 h.
The above ligand products can be used directly for E. coli transformation or electrophoresis. Before transformation or electroporation, the microcentrifuge tube containing the ligand should be heated at 70℃ for 10~15 min to re-melt the solidified gel.
Typically, 1-5 ul of ligation product can be used to transform chemically prepared cells, whereas only 0.1-1.0 ul of ligation product is required for the more efficient electroporation transformation. The use of larger volumes for electroporation increases the concentration of solutes to the point where the onset of arcing is difficult to predict.
The source of this experiment is "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.