buffer without MgCl 2 , 1.5 μL of 25 mM MgCl 2 , 0.5 μL of
25 mM dNTPs, 0.6 μL of 10 μM oligo 1 (TnK1 or TnK2),
0.2 μL of 100 μM oligo 2 (Deg1 or Deg2), 0.25 μL of Taq
DNA polymerase, 17 μL of nuclease-free water, 2 μL of template DNA (40 ng total) (see Note 41).
4. Run round 1 with the following reaction times and temperatures: Step 1, denature at 95
C for 5 min; step 2, denature at
95
C for 15 s, anneal at 45
C for 1 min, extend at 72
C for
2 min, and repeat step 2 39 additional times; and step 3, extend
at 72
C for 10 min (see Note 42).
5. Set up a mix for round 2 of nested PCR by using semi-random
primers with the following reagents and quantities per reaction
tube (total volume ¼ 25 μL): 2.5 μL of 10Â Taq polymerase
buffer without MgCl 2 , 1.5 μL of 25 mM MgCl 2 , 0.5 μL of
25 mM dNTPs, 0.25 μL of 100 μM oligo 3 (TnKN1 or
TnKN2), 0.25 μL of 100 μM oligo 4 (Tag), 0.2 μL of Taq
DNA polymerase, 19 μL of nuclease-free water, 0.8 μL of PCR
round 1 DNA (see Note 43).
6. Run round 2 with the following reaction times and temperatures: Step 1, denature at 95
C for 5 min; step 2, denature at
95
C for 15 s, anneal at 53
C for 30 s, extend at 72
C for
2 min, and repeat step 2 34 additional times; and step 3, extend
at 72
C for 10 min.
7. Run nested PCR products on a 1Â TBE gel which will visualize
multiple bands typically ranging from 100 to 1000 bp in length
(Fig. 7) (see Notes 44 and 45).
Fig. 7 Example of semi-random primer nested results after round 2. Lanes 1–5:
5 L. biflexa Tn mutants. Lane 6: negative control. For this experiment, round
1 primers used were TnK1 and Deg1. Round 2 primers used were TnKN1 and
Tag. Electrophoresis completed using a 0.8Â TBE gel adulterated with RedSafe,
visualized with a Bio-Rad Gel Doc EZ System
Random Transposon Mutant Library
87