Cloning

Cloning protocols - http://www.its.caltech.edu/~bjorker/Protocols/cloning%20protocols%20by%20Astrid.pdf Bjorkman Lab. Cloning protocols and tips by Astrid PCR: Primer design: I normally make the complementary part of my primers 18 bp long and make sure that there are enough bases behind a restriction site (NEB 249) so you can do a direct restriction on the PCR product. Set up the following reaction mixture and mix well: - [Read more]

Cloning Tips - http://www.its.caltech.edu/~bjorker/Protocols/How_to_label_DNA_tubes.pdf Cloning Tips. REQUIREMENTS FOR LABELING TUBES OF DNA IN THE BJORKLAB (Why?....Regrettable absence of psychic ability among the Bjorkpeople... Weeks (literally weeks, I'm not kidding) spent trying to decipher the mystic markings on tubes of DNA left by previous lab members and/or redoing things due to incorrect decoding... Grouchiness induced by this waste of time... Additional grouchiness induced by - [Read more]

Cloning with Invitrogen TOPO TA Cloning kit - http://people.bu.edu/pbarber/Web%20Protocols/Cloning%20PCR%20fragments.pdf Barber Lab Cloning with Invitrogen TOPO TA Cloning kit This procedure will insert your PCR fragment into a suitable vector in this case (pCR2.1- TOPO). This vector has antibiotic resistance (Ampicillian and Kanamycin) as well as having a gene for LacZ production. The successful insertion of a fragment into the ligation site will interrupt the LacZ gene and when grown on X-gal plates will result in white colonies. - [Read more]

Making LB Agar Plates - http://people.bu.edu/pbarber/Web%20Protocols/LB%20Agar%20Plates.pdf Making LB Agar Plates Batch makes about 40 plates. Making the LB Agar 1. Add 250 mL of dH2O to a graduated cyclindar. 2. Weigh out 20g of premix LB Agar powder (VWR DF0445-17) or: 3. 5.0 g tryptone 4. 2.5 g yeast extract - [Read more]

Preparation of Electrocompetent E. coli - http://www.its.caltech.edu/~bjorker/Protocols/Prep_of_electocomp_cells.pdf Bjorkman Lab. Preparation of Electrocompetent E. coli (i.e. DH5 ) revised 2/24/96 Before starting procedure, prepare/chill the following: 1 L 2XYT media (no antibiotics!), store at RT 1 L chilled autoclaved dH20, stored in 4° cold room. 100ml chilled 10% glycerol / dH20 solution, store at 4°C. 4 Corning 250ml pointed bottles (w/ orange caps) with white adapters chilled at 4°C. - [Read more]

Site-directed Mutagenesis Protocol - http://www.its.caltech.edu/~bjorker/Protocols/Site-directed_mutagns_pro.pdf Bjorkman Lab. Dut/ung in vitro Site-directed Mutagenesis Protocol pg. 1 (originally written by M. Davis and P. Bjorkman) revised: 12/20/95 Favored E. coli strains for site-directed mutagenesis using uracil replacement: RZ1032: dutts, ung, tetr Bw313: dut: deoxy uracil transphosphatase (enzyme required for formation of thymidine precursor) ung: uracil-DNA-glycosylyase (enzyme which normally will proof and replace uracil in DNA) - [Read more]

Sub-Cloning - http://www.its.caltech.edu/~bjorker/Protocols/PJB%27s_cloning_hints.pdf Sub-Cloning. PJB’s hints for sub-cloning: 1) Prior to any ligation reaction, you should always run one gel in which purified insert and cut backbone are run side by side, preferably beside a known amount of cut DNA. You can then use this to estimate how much backbone and insert to use in your ligation reaction. In general, a large excess of insert over backbone works well, but you should - [Read more]

Subcloning procedures - http://www.its.caltech.edu/~bjorker/Protocols/Martin_subcloning%20procd.pdf Bjorkman Lab. Subcloning procedures These are in as modular a format as possible my preferred methods for subcloning. They are not in anyway the only way to proceed but if they are followed with precision I will vouch for their efficacy. General notes of strategy (possibly the most IMPORTANT section) We subclone to tailor reagents to experiments. This simple thought must govern all - [Read more]