Berglund:Stratagene Quickchange

Quick Change Protocol-mBBP 1-360 fixing a glycine mutation

Forward and Reverse Mutagenic primers: Both primers should contain the mutations of interest 25-45 bases in length Tm should be greater than or equal to 78°C Calculate for these primers with mutations: Tm=81.5+0.41(%GC) – 675/N	(N= # of all bases EXCEPT the mutation bases) The mutation should be in the middle of the primer with 10-15 bases of correct sequence on either side Primers should have a GC content of 40% or more and should terminate in one or more C’s or G’s. Primers must be cleaned using a polyacrylamide gel, otherwise mutation efficiency decreases. Primer content during PCR must be in high excess of the template (changing template conc may help)

STEP 1: designing the primers Want to fix the glycine mutation in mBBP 1-360 plasmid in pgex ATGGCGACCGGAGCGAACGCCACGCCGTTGGACTTCCCAAGTAAGAAGCGGAAGAGGAGCCGCTGGAACC AAGACACAATGGAACAGAAGACAGTGATTCCAGGAATGCCTACAGTTATTCCCCCTGGACTTACTCGAGA ACAAGAAAGAGCTTATATAGTGCAACTGCAGATAGAAGACCTGACTCGTAAACTGCGCACAGGAGACCTG GGCATCCCCCCTAACCCTGAGGACAGGTCCCCTTCCCCTGAGCCCATCTACAATAGCGAGGGGAAGCGGC TTAACACCCGAGAGTTCCGCACCCGCAAAAAGCTGGAAGAGGAGCGGCACAACCTCATCACAGAGATGGT… W/A= TGG to GCG GC= 55% TM=73°C

AemBBP1-360gly F or R 5’ CCAAGTAAGAAGCGGAAGAGGAGCCGCTGGAACCAAGACA Forward 5’ TGTCTTGGTTCCAGCGGCTCCTCTTCCGCTTCTTACTTGG Reverse

STEP 2: PCR with Advantage taq, 16 cycles PCR reaction 38µl	water 5µl	thermalace buffer 1µl	50xdNTP 2µl	forward 10mM 2µl	reverse 10mM 1µl	template 1µl	advantage taq

program: 95°C 30sec 95°C 30sec, 55°C 1 min, 68°C 6min (16 cycles total) 37°C infinity

STEP 3 Dpn 1 digestion (digestion of methylated DNA) Add 1µl of Dpn1 enzyme to each reaction. Mix well Incubate each reaction for 1 hr at 37°C in the thermocycler with the hot bonnet to digest the template

STEP 4 ethanol ppt, resuspend into 8 µl, ligate overnight

STEP 5: transformation into tg1 cells, use 5µl

STEP 6: sequencing with pGEX3 primers, cut inserts to see if the insert is still the proper size, may need an internal primer