Virtualab Protocols (Chang Bioscience)
PCR products using Taq DNA polymerase may have an extra nucleotide,
most likely A, at their 3'-ends because of the transferase activity of
Taq DNA polymerase. Other PCR polymerases may not have the transferase
activity. PCR products with a 3' A can be ligated to a vector with 3' T.
1. To make a vector with 3' T (T-Vector), digest 5 mg
vector DNA with an restriction enzyme that produces blunt ends. Check
with agarose gel electrophoresis to make sure the digestion is complete.
Add a little fresh enzyme and continue digestion if necessary.
2. After completion of the digestion, cool the digestion mix on ice.
Remove protein with phenol:chloroform:isoamyl alchol (25:24:1) extraction,
and precipitate DNA with ethanol.
3. After wash and dry, resuspend the pellet in 172 ml
of ddH2O. Make the following mix:
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Digested DNA in ddH2O (5 mg)
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172 ml
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10X PCR Buffer
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20 ml
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10 mM dTTP
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4 ml
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Taq DNA polymerase (10 units)
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4 ml
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4. Incubate at 72 °C for 1 hour to make the T-Vector. Incubate either in a 72 °C heat block
or program PCR thermocycler to run at 72 °C for 1 hour.
5. Add 1 ml to a ligation mix to test
for self-ligation. Transform E. Coli cells with 1 ml
of unligated T-Vector, and self-ligation products. Few clonies should form
for both.
6. For cloning of PCR products, ligate 1-5 ml
of PCR products to 1 ml of T-Vector.
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