Muratore:Materials/Primers

BSA-NheI-5'
5'- CCC ACA AAA CTA TGG CTA GCA AGT GGG TGA CT -3'

PCR primer to insert BSA into IMPACT vectors

Tm = 69.5°C

Length = 32, LenPr = 15, MaxMM = 4

Priming to pCMV-SPORT6+BSA: 5'-CCCACAAAACTATGGCTAGCAAGTGGGTGACT-3' CCCACAAAACTGGCACAATGAAGTGGGTGACT priming site at position 161 forward Tm = 52.5°C. Tpcr = 51.3°C.

BSA-SapI-3'
5'- TGG TTG GCT CTT CTA CGG GCT AAG GCT GT -3'

reverse PCR primer to insert BSA into IMPACT vectors

Tm = 71°C

Length = 29, LenPr = 15, MaxMM = 4

Priming to pCMV-SPORT6+BSA: 5'-TGGTTGGCTCTTCTACGGGCTAAGGCTGT-3' TGGTTGTGTCGTGTTTAGGCTAAGGCTGT priming site at position 1956 reverse Tm = 41.4°C. Tpcr = 47.9°C.

ins_His_after_CBD
5'- CCT TGT GGC AGC TTC AAC ACC ACC ATC ATC ATC ACC ACC ACT GAC TGC AGG AAG GG -3'

Quikchange primer to insert His8 tag at C-term of Chitin binding domain tag in IMPACT vectors

Tm = 80.4°C

Length = 56, LenPr = 8, MaxMM = 2

Correct priming to pTXB1: 5'-CCTTGTGGCAGCTTCAACACCACCATCATCATCACCACCACTGACTGCAGGAAGGG-3' GATGGGAACCATCCAACGTTCCTGCCTTGTGGCAGCTTCAATGACTGCAGGAAGGG priming site at position 6570 forward Tm = -0.8°C. Tpcr = 35.3°C.

Side-reaction priming to pTXB1: 5'-CCTTGTGGCAGCTTCAACACCACCATCATCATCACCACCACTGACTGCAGGAAGGG-3' AGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGG priming site at position 804 reverse

Tm = 3.7°C. Tpcr = 36.6°C.

Rins_His_after_CBD
5'- CCC TTC CTG CAG TCA GTG GTG GTG ATG ATG ATG GTG GTG TTG AAG CTG CCA CAA GG -3'

reverse Quikchange primer to insert His8 tag at C-term of Chitin binding domain tag in IMPACT vectors

Tm = 80.4°C

Length = 56, LenPr = 10, MaxMM = 2

Correct priming to pTXB1: 5'-CCCTTCCTGCAGTCAGTGGTGGTGATGATGATGGTGGTGTTGAAGCTGCCACAAGG-3' TCGGGCTTTGTTAGCAGCCGGATCCCCTTCCTGCAGTCATTGAAGCTGCCACAAGG priming site at position 6539 revers Tm = 20.1°C. Tpcr = 41.6°C.

Side-reaction priming to pTXB1: 5'-CCCTTCCTGCAGTCAGTGGTGGTGATGATGATGGTGGTGTTGAAGCTGCCACAAGG-3' GCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGG priming site at position 4620 forward Tm = -3.6°C. Tpcr = 34.4°C.

Priming and melting/PCR temperatures are calculated using | pDRAW