The following section describes which primers you will need to design for performing targeted genome modifications (deletion and in locus overexpression) in fungi by USER Friendly cloning

You will need primers for 1) construction of the vector, 2) verification of the vector and 3) verification of the genetically modified strain. Instruction and tips for the three different primer types can be found in the following sections.

(Need to obtain the Agrobacterium tumefaciens plasmids with USER cloning sites click here)

1. Primers for construction of the required vector:

1. Design gene specific primers using a suitable program.
2. Append the overhangs, as described below.
3. When you order the primers, it is important that you specify that the primers contain a non-standard base: 2-Deoxyuridine which is the DNA version of uracil (sometimes this is specified as a modification depending of the vendor).

TIP: Save time and money - If you are going to do both targeted replacement and in locus overexpression of the same gene, reuse the primer 1 and 2 for both tasks (see below).

For replacement vectors  

Insert 1 (promoter region of target gene)     

Primer 1:        5’- GGTCTTAAU-Gene specific sequence

Primer 2:        5’- GGCATTAAU-Gene specific sequence

Insert 2 (terminator region of target gene)

Primer 3:        5’- GGACTTAAU-Gene specific sequence

Primer 4:        5’- GGGTTTAAU-Gene specific sequence

   (Download Excel template for the in silico implementation of the vector construction)

I normally use the following standard primers to verify all constructs by PCR in combination with the terminal primers used for construction of the vector (remember to use Taq DNA polymerase as the construction primers include uracil).

I also use these primers for sequencing of the constructs before proceeding to ATMT.

For deletion strains I normally use four primer pairs:

1. Testing for the selection marker*
2. Testing for loss of the target gene (annealing at the center of the coding sequence)
3. Testing for correct cross-over at the left flank (annealing upstream of the homologous recombination sequence, in combinations with RF-1, RF-2 or RF-3 depending of the construct)
4. Testing for correct cross-over at the right flank (annealing downstream of the homologous recombination sequence, in combinations with RF-1, RF-2 or RF-3 depending of the construct)