DNA polymerase must have a primer when performing DNA polymerization, because it can only use the 3'-OH provided by the primer as the starting point for extension work, and cannot re-synthesize new DNA in situ. (de novo synthesis). In general experiments, artificially synthesized oligonucleotides are often used as primers for DNA polymerization. Although nucleic acid primers can be designed based on known sequences, random primers can also be used. The so-called opportunity primer is a sequence of opportunity, without special design, and the most widely used at present is an oligonucleotide mixture of six to eight nucleotides synthesized by an automated machine; if the reaction is in progress Any opportunity primer can be combined with the template DNA to guide the DNA polymerization reaction. The random priming method is used for nucleic acid calibration. The Klenow fragment (large fragment of E. coli DNA polymerase I) is guided by the opportunity primer to carry out DNA polymerization reaction, and [Î±-32P] dNTP or DIG-11-dUTP is added And other calibration substances. In order to avoid random priming also occurring in the DNA of the carrier, it is best not to use the plastid DNA as a template for the calibration reaction, but to remove the target DNA from the carrier in advance.
Equipment: micro centrifuge; boiling water bath and ice bath
Template DNA (pBlueGus' BamHI-HindIII DNA fragment, to be provided by the teaching assistant)
0.2 M EDTA, pH 8.0
4 M LiCl
Random priming kit (Roche):
Hexanucleotide mix (random primer)
dNTP mixture (1 mM dATP, 1 mM dCTP, 1 mM dGTP, 0.65 mM dTTP, 0.35
mM DIG-11-dUTP, pH 7.5)
Klenow enzyme (2 U / Î¼L)
TE (10 mM Tris-Cl, pH 8.0; 1 mM EDTA)
â—† The following reaction conditions and steps refer to the product manual provided by the manufacturer of random priming kit.
1) Take 100 ng template DNA, add appropriate amount of deionized water, and make up the volume to 15 Î¼L.
2) Heat in boiling water for 10 min.
â—† Please remember to fix the cap part of the microcentrifuge tube with a clip to prevent the cap from bursting and water entering during the heating process!
3) After heating, quickly move the microcentrifuge tube to an ice bath and let it stand for a few minutes.
4) After centrifuging for a few seconds, add the following three reagents in sequence:
2 Î¼L hexanucleotide mix
2 Î¼L dNTP mixture
1 Î¼L Klenow enzyme
5) Flick the centrifuge tube with your finger to mix evenly.
6) After short centrifugation, place in a 37 Â° C constant temperature water bath for 2 h.
â—† The reaction time should be at least 1 h, and the longest reaction time is overnight.
7) After the action is completed, add 2 Î¼L of 0.2 M EDTA to terminate the DNA polymerization reaction.
8) Add 2.5 Î¼L of 4 M LiCl and 75 Î¼L of pure alcohol, mix well, and set at -20 â„ƒ overnight.
The next day:
1) Centrifuge at 13,000 rpm and 4 Â° C for 15 min.
2) Decant the supernatant, add 50 Î¼L of 70% alcohol, and centrifuge for 5 min.
3) Decant the supernatant and air dry the DNA.
4) Finally, dissolve the DNA with 50 Î¼L TE.
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