1. Molecular cloning overview Steps to prepare vector 1.12-16 hour overnight culture 2.Minipreps (day immediately following O/N) (use Qiaprep spin miniprep kit) Measure DNA conc. 3.Large-scale restriction enzyme digest (Preferably same day as minipreps) 4.Large-well agarose gel electrophoresis, ensure bands are expected sizes, cut out correct band from gel 5.DNA extraction from agarose (use Qiaquick gel extraction kit) Measure DNA conc. 6.CIP (calf intestinal phosphatase) digestion 7.DNA precipitation (with phenol chloroform, etc) Measure DNA conc. Overnight culture may be started from a frozen cell stock, or colony from agar plate Start several 5 mL cultures to make sure there is enough material at the final step (5 or more) After measuring the DNA concentration to ensure minipreps were all successful, combine all the DNA into one microcentrifuge tube for the digest For pET28b GroES7, the larger band will be at 7500 base pairs The digest will remove one subunit at 300 base pairs DNA precipitation procedure takes 2 days Afterwards, DNA is ready for ligation

2. Molecular cloning overview Steps to prepare insert 1.PCR amplification2.Agarose gel, confirm product is correct size 3.TOPO reaction, use 3 uL of PCR product 4.Transformation into super- competent cells, plate onto spectinomycin agar (prefarably same day as TOPO reaction) 5.12-16 hour overnight cultures 6.Minipreps (day immediately following O/N) (use Qiaprep spin miniprep kit) Measure DNA conc. 7.Test digests of miniprep DNAs For GroES7 cloning, use GroES single as the template DNA for PCR, product should be 300 base pairs Start 4-6 cultures from agar plate Use 1 uL of DNA if concentration is over 200 ng/uL, otherwise use 3 uL of DNA, final reaction volume 10 uL 8.Agarose gel of test digests, confirm bands are correct size Add 2 uL of 6X DNA loading dye to each sample, load all 12 uL onto gel TOPO vector is 2.8 kb TOPO vector is cut in half by XhoI enzyme, makes one strong band at 1.4 kb

3. Molecular cloning overview Steps to prepare insert, continued 9.Large-scale digest of one correct clone 10.Large-well agarose gel electrophoresis, ensure bands are expected sizes, cut out correct band from gel 10.DNA extraction from agarose (use Qiaquick gel extraction kit) Measure DNA conc. DNA is ready for ligation

4. Molecular cloning overview Ligation 1.Set up ligation reactions, let incubate at room temperature 8+ hours 2.Transform 5 uL of each ligation reaction into super-competent cells 3. Select 2-6 colonies from each plate, set up 12-16 hour overnight cultures 4. Minipreps (day immediately following O/N) (use Qiaprep spin miniprep kit) Measure DNA conc. If control plates have many colonies, screen more clones 5.Test digests of miniprep DNAs Use 1 uL of DNA if concentration is over 200 ng/uL, otherwise use 3 uL of DNA, final reaction volume 10 uL 6.Agarose gel of test digests, confirm bands are correct size Add 2 uL of 6X DNA loading dye to each sample, load all 12 uL onto gel 7.If gel looks good, set up sequencing reactions for correct clones to confirm Set up and run PCR program 1 before the date of sequencing plate (not any farther in advance) Drop off sequencing reactions by 3pm on date of sequencing plate

5. Molecular cloning overview Steps for Quik-Change mutagenesis (site-directed mutagenesis, point mutation) 1.PCR amplification3. Agarose gel, confirm product is correct size 2. Add 1 uL of DpnI, digest for 1 hour 4.Transform into super-competent cells, 5.12-16 hour overnight cultures 6.Minipreps (day immediately following O/N) (use Qiaprep spin miniprep kit) Measure DNA conc. Start 4-6 cultures from agar plate 7.Set up sequencing reactions for clones to confirm Set up and run PCR program 1 before the date of sequencing plate (not any farther in advance) Drop off sequencing reactions by 3pm on date of sequencing plate Do not let digest go for over 1 hour

6. Molecular cloning overview This handbook is a guide- your experiments may be different, depending on what DNAs are used, and the final outcome of samples Last updated 2012 03 19 Melissa Illingworth