Theoretical and Experimental DNA Computation (Natural ...

5.8 Experimental Investigations 125
Table 5.3. Summary of experimental objectives
No. Summary of experimental objective
1 Resuspension of oligos
2 Creation of initial library
3 Amplification of initial library
4 Purification of product of Experiment 3
5 Check to ensure correct hybridization
6 Test of specificity of Experiment 5
7–8 Cloning and sequencing to ensure correct library construction
9 Removal experiment 1
10 Amplification and purification of test library subset
11 Removal experiment 2
12 Removal experiment 3
13 Test of ability of Sau3A to digest dsDNA
14–15 Control experiments using MboI rather than Sau3A
16 Removal experiment 4
17 Removal experiment 5
18 Removal experiment 6
19 Removal experiment using Klenow (7)
20 Removal experiment 8
21 Removal experiment 9
22 Test of multiple removals
23–24 Oligo redesign
25 New initial library construction
26–29 New initial library amplification
30–31 PCR control experiments for new library
32 Removal experiment 10
Experiment 4. Purification of v1–v8 PCR product
The 10 best samples producing bands in Experiment 3 were pooled, and gel
purified on 2% agarose. The ∼200 b.p. fragment was extracted using the Qiagen
gel extraction kit. The PCR product was ligated into a T cloning vector
and used to transform stored competent bacteria. Several thousand clones
were produced. 12 were grown, miniprepped, and sequenced (Experiments 7
and 8).
Experiment 5. PCR between v2 and v8
The aim was to check that all three colorings of v2 were present in the amplified
chain produced in Experiment 3. This was done simply by using v8
and either red, green, or blue specific v2 primers in a standard detection PCR
reaction.