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September 29th

Touchdown PCR

Because the new primers have long sequences that are not supposed to bind to the target DNA (the pre- and suffix for forward and reverse primer, respectively), a low annealing temperature could lead to a lot of aspecific product formation. To suppress this, a touchdown PCR was performed with the new primers on the PCR products of atoB, idi and ispA and genomic DNA of E. coli. In both cases 60 ng of template DNA and the platinum Pfx enzyme was used. The PCR program looked like this:

1. 5' @ 94°C
2. 1' @ 94°C
3. 1' @ 65°C --> temperature is lowered with 0.5°C per cycle
4. 3' @ 72°C
5. go to 2, 20 cycles in total
6. 1' @ 94°C
7. 1' @ 94°C
8. 3' @ 72°C
9. go to 6, 20 cycles in total
10. 7' @ 72°C
11. ∞ @ 10°C

The products were put on gel:

Circled areas indicate cut bands, after the picture was taken some extra agarose in the idi lane was removed. Expected sizes were for atoB ~1200bp, idi ~580bp and ispA ~975bp

As can be seen, the PCR did not succeed for the genomic DNA, but it did work on the PCR products. The reason for this is probably that although there is an even amount of template DNA in both cases, there is of course a lot more specific DNA binding spots in the PCR product. The gel bands were cut (see figure) and DNA isolated using a Qiagen kit. The 260/280 was around 1.7 and DNA content around 21 ng/ul for all products (atoB, idi and ispA) according to NanoDrop.

Growing for midiprep

K115022 and K115027 are incubated o/n @ 37 degrees Celcius.