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文献和实验AFLP: not only for fingerprinting, but for positional cloning
-adapter 1.0 µL MseI-adapter 1.0 µL 10 mM ATP 4.0 µL 5x-OPA+-BSA 1 U T4 ligase (Pharmacia) Q.S. to 10 µL w/ dH20 - Incubate @ 37° C for 3 hrs to O/N 2.0 Pre-amplification of template DNA [Note 1: This pre-amplification step helps to "clean
buffer.In control digests, include 10µg genomic DNA plus 10-5, 10-6, 10-7µg of plasmid containing insert complementary to probe.Load on far side of gel away from test samples.As usual, include size standards.Use a 0.7% agarose mid-size gel in 0.5 x TBE
Cloning via Restriction Digests
restriction enzymes for your insert and vector, and determine the appropriate reaction buffers. Combine the following in a microfuge tube (30 μL total volume): 2 μg DNA 1 μL Each Restriction Enzyme 3 μL 10x Buffer 3 μL 10
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