Lab Post #2 (8/29/16)

1 out of every 10 million cells has a double strand break within our target genes so that we can remove SAK1 and TOS3.

To identify successfully mutated cells we will be using antibiotic resistance tags.

We will be using PCR (denaturing the DNA with high temperatures and utilizing primers to allow polymerase to complete the sequence) to amplify template DNA of yeast mutation. These DNA strands will be used later create a new mutant yeast strain.

Designed Primers:

Found with the help of http://www.yeastgenome.org/cgi-bin/seqTools using 500 flanking pairs pre and post SAK1/TOS3 and https://www.idtdna.com/calc/analyzer to analyze the bases (aiming for 62C and 20-30 bases)

SAK1

Initial:

AGC GGC TTG TTC CTT CGT TCC C

62.3C

Bases: 22

Final:

TTC TCA GGG TGA GAC GAA GAA ACG AAA AGT

61.1C

Bases:30

TOS3

Initial:

CCT TGA AAT TGC TGC GCA CAT ATT CTG CAT

61.7 C

Bases: 30

Final:

TGA AAT CCA GCT CTT TTA TAC CTG GAT GGG

59.8 C

Bases: 30

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