Practical course 5: Inverse PCR - deletion of gene segments

Insertion of deletions in practice: primer processing and PCR

Purification of the oligonucleotide primers

Before performing the PCR, the primer must first be cleaned and the concentration optimally adjusted. The template / primer ratio is very important for a successful PCR - if there are too many template molecules in the PCR mixture, the yield of PCR product decreases, while a primer concentration that is too high promotes the formation of unspecific PCR products.

The oligonucleotides are usually supplied in ammoniacal solution and purified by DNA precipitation:

  • 360 µL oligonucleotide solution
  • + 36 µL 3 molar NaOAc solution
  • + 1.2 mL cold ethanol (100%, denatured)
  • 12 h (at least 3 h) at -20 ° C
  • 15-30 min in the biofuge at 13,000 rpm1) centrifuge
  • wash with 200 µL cold, 80% ethanol
  • Pellet in 50 µL redist. H2O record

Determination of the primer concentration

To determine the UV adsorption (260 nm), 2% of the mixture is diluted 1:60 in a 50 µL quartz cuvette. The amount of oligonucleotide in the given 50 µL can be calculated as follows: One OD260 of 1 corresponds to 20 µg / mL oligonucleotides.

OD260 x dilution factor x 20 = oligonucleotide concentration in µg / mL

The average molecular mass of a dNTP is 350 Da; this number has to be multiplied by the length of the oligonucleotide in order to calculate the optimal primer concentration, which should be 0.1-1 µM.

Performing the PCR

The following mixture is pipetted into a 0.5 mL PCR tube:

Tab. 1
Composition of the approach
5 µL (0.5-1 µM)Primer pUC19-1
5 µL (0.5-1 µM)Primer pUC19-2
5 µL20 x buffer
6 µL25 mM MgCl2
1.5 µLTfl DNA polymerase
0.5 µLpuc19-DNA (template)
3 µLNucleoside triphosphate mix
= 26 µL+ 74 µL redist. H2O

The batch is mixed and then placed in the thermal cycler. This is programmed as follows:

Tab. 2
Programming the thermal cycler
occurrenceReaction conditions
Denaturation90 ° C, 1 min
Annealing54 ° C, 1 min
Elongation74 ° C, 4 min, 20 cycles
Final synthesis72 ° C, 10 min

After the program has ended (approx. 1.5 h), the PCR batch is removed. In order to smooth the ends, i.e. in order to possibly complete incomplete syntheses, the Klenow fragment is now used for theE. coli- DNA polymerase I treats:

Tab. 3
Approach to follow-up treatment
100 µLPCR approach
+ 2 µLdNTPs
+ 1 µLKlenow polymerase

The reaction takes place for 20 min at room temperature.