6. Simultaneous digestion of the pUC vector with both enzymes in the presence of 3 units of Shrimp Alkaline Phosphatase (Amersham BioSciences) in potassium glutamate buffer (KGB) usually provides the best material for cloning. Sequential extended digests should be avoided due to exonuclease activity present in many lots of enzyme. BamH I and Hind III must have low levels of exonuclease activity when used under these conditions. BamH I and Hind III were chosen because they are the most compatible double digest restriction enzymes available for cloning into the polylinker. Star activity due to excessive enzyme concentration must be avoided.[4]
7. KGB may cause some distortion of fragments < 800 basepairs on agarose gels of < 1%.[5]
8. Avoid using ligation buffer that shows the slightest trace of precipitation.
9. UV-generated damage to DNA stained with ethidium bromide reduces the transformation efficiency. One minute of exposure to 312 nm UV can reduce the transformation efficiency by > 90%.[6] [7] UV-damaged DNA products include cyclobutane pyrimidine dimer (T-T), 5-thyminyl-5,6-dihydrothymine, 4,6-diamino-5-formamidopyrimidine, 5-hydroxy-5,6-dihydrothymine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 6-hydroxy-5,6-dihydrocytosine and 8,8-adenine dehydrodimers (from poly(dA). Guanosine is reported to protect DNA from damage by UV transillumination. 1 mM guanosine added to the gel and the running buffer “increased the yield of clones by a factor of about 400, compared with conventionally prepared, unprotected DNA” exposed to 312 nm UV.[8]
10. SYBR Gold is more sensitive than ethidium bromide and can be removed by ethanol precipitation.
11. Since 66 µM dATP inhibits T4 DNA ligase by 60%[9], the PCR amplified insert should be purified before being used in the ligation reaction.
12. To estimate the frequency of colonies with the correct insert, 10 µL samples from 10 to 20 cultures can be PCR amplified according to a procedure for amplification of bacterial colonies,[10] using the forward and reverse sequencing primers. Colony PCR initiated from transformation reactions can give rise to false positives if the primers anneal to the insert sequence due to excess DNA insert from the ligation reaction that is spread as part of the transformation reaction onto bacterial plates.[11] Typical ligation protocols utilize 5 pmol of insert PCR product in a 20 µL reaction (0.25 pmol/µL) with 0.5 pmols of vector. Assuming that all the vectors acquire a single copy of insert, 4.5 pmols (0.225 pmol/µL) of excess insert remain in the ligation reaction. 2 µL of a 1:5 dilution of the ligation reaction (0.09 pmol) is electroporated and diluted 1:9 with SOC media (0.01 pmol/µL). If 100 µL (1.0 pmol) is spread on a 55.4 cm2 plate (0.018 pmol/mm2) and a 4 mm2 agar pick is used, then 0.072 pmols (4.35 x 1010 molecules) of background insert are available for a 100 µL colony PCR assay. To avoid this problem, at least one primer should anneal to the vector. Use of the Forward and Reverse Sequencing primers is recommended, as this allows the determination of double inserts or vector-only colonies. A negative control consisting of vector-only is also recommended for purposes of band identification.
13. Transfer 10 µL of overnight cell culture to a 650 µL microfuge tube containing 500 µL Type I water. Heat the diluted culture at 95°C for 5-10 minutes to lyse the cells, destroy proteinases and release denatured plasmid DNA. Temperature is critical; variation due to inadequate heating of differing volumes of water squirted into the heating block or a heat block that is below about 93°C will result in the failure of this technique. Use a thermistor to confirm the 95°C temperature. Microfuge at room temperature for 2-5 minutes. If a cell pellet is visible after this treatment, you added too many cells or the heating method failed to reach the correct temperature.
14. Combine 2 µL of 10x AmpliTaq buffer II, 0.4 µL of 10 mM dNTPs, 1 µL @ 10 µM FSP and RSP, 5 µL of cell extract, 0.5 µL of 5 Unit/mL AmpliTaq and Type I water to a final volume of 20 µL. Overlay the reactions with 20 mL of silicone oil (if required) and incubate in a Perkin Elmer Thermal Cycler 480, at 93ºC for 3 min, followed by 20-30 cycles of 93ºC for 30 sec, 55ºC for 60 sec and 72ºC for 1 min. The program for the Perkin Elmer 9600 is 93ºC for 3 min, followed by 20-30 cycles of 93ºC for 30 sec, 55ºC for 15 sec and 72ºC for 1 min. Analysis of 10 µL of each reaction with the GenSura 20/100 bp ladder on an 8% native acrylamide gel stained with ethidium bromide or SYBR Gold will determine which colonies contain single inserts of the correct size. If all of the analytical samples have a single insert, the remainder of the clones are assumed to have inserts also and are scheduled for sequencing. In the event of any vector-only or primer-dimer clones, all of the samples should be checked before sequencing.
1. Scrupulously avoid detergent contamination of all containers used in this process and carry out all processes aseptically.
2. Luria Broth (LB): combine 10 g of Bacto tryptone (Difco, Detroit, MI), 5 g of Bacto yeast extract (Difco) and 5 g of NaCl with 800 mL of water and adjust the pH to 7.0. Adjust the media volume to 1 liter before autoclaving. Note that you will need 4 x 500 mLs of LB in order to prepare competent cells. Store at room temperature. Do not substitute LB prepared with 10 g of NaCl, as arcing may result during electroporation.
3. 100 mL ice cold 10% glycerol: Combine 10 mL of enzyme grade glycerol with 90 mL of Type I water. Filter sterilize through prerinsed filtration units. Autoclaving glycerol can reduce the transformation efficiency of the competent cells. Glycerol will start to decompose to ethylene aldehyde at its boiling point of 182°C.
4. Four liters of ice cold filter sterilized Type I water
5. Chilled JA10 rotor or equivalent
6. Six ice cold 500 mL sterile centrifuge bottles
7. 80-100 ice cold sterile screw-cap cryogenic vials (labeled)
8. Electroporation apparatus and ice cold 1 mm gap cuvettes
9. SOC contains twice the Bacto tryptone of LB to provide amino acids for rapid repair of cell walls damaged by the generation of competent cells and electroporation. NaCl is used to maintain isotonicity and glucose is provided for an energy source. SOC consists of 2% Bacto tryptone, 0.5% Bacto yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4 and 20 mM glucose. Combine 20 g of Bacto tryptone, 5 g of Bacto yeast extract, 10 mL of 1 N NaCl, 2.5 mL of 1 M KCl, 5 mL 1 M MgCl2, 5 mL 1 M MgSO4, 10 mL of 2 M glucose and 800 mL of Type I water. Adjust the pH to 7.0 ± 0.1, adjust the volume to 1 liter and filter sterilize.
10. 100 mg/mL sodium ampicillin: dissolve 5 g of sodium ampicillin in Type I water and adjust to a final volume of 50 mL. Filter sterilize and aliquot for storage at -20°C. A final concentration of 100 µg/mL is achieved by adding 1 mL/liter of media. Carbenicillin may be substituted at equal concentrations in order to reduce the number of satellite colonies.
11. 200 mg/mL methicillin: dissolve 500 mg of methicillin (Research Diagnostics, Flanders, NJ) in 2.5 mL of Type I water. Filter sterilize and aliquot for storage at -20°C. A final concentration of 200 µg/mL is achieved by adding 1 mL/liter of media or agar stock. Ampicillin selection of high copy number plasmids such as pUC is more effective when methicillin is combined with ampicillin, since satellite colony formation is strongly inhibited. Do not use methicillin with wild type copy number plasmids unless you are selecting for high copy number mutations.
12. Triphenol tetrazolium chloride (TTC) is prepared as a 1000x stock at 10 mg/mL and can be added to a final concentration of 10 µg/mL. TTC is used to plate out the transformation reactions. Under these conditions, all of the colonies will have red cores, making detection of side-by-side colonies easier.
13. 2% (w/v) 5-bromo-4-chloro-3-indoyl-B-D-galactoside (X-gal). In a glass container, dissolve 1 g X-gal in n,n-dimethylformamide to a final volume of 50 mL. Store at -20°C. The final concentration in plates is 0.4 mg/mL. X-Gal/IPTG plates are used for re-streaking the TTC colonies prior to liquid culture to eliminate co-transformation of library sequences.
14. 100 mM isopropylthiogalactoside (IPTG). Dissolve 1 g IPTG in Type I water to a final volume of 42 mL. If no particulate material remains, aliquot into microfuge tubes and store at -20°C. The final concentration in plates 0.2 mg/mL.
15. 2xYT medium consists of 1.6% Bacto tryptone, 1% Bacto yeast extract, 10 mM NaCl Combine 16 g of Bacto tryptone, 10 g of Bacto yeast extract, 5 g of NaCl in 800 mL Type I water. Adjust the pH to 7.0 and bring the volume to 1 liter. Aliquot to 100 mL and autoclave.