Gel extraction

QIAquick®Gel Extraction Kit

Notes before starting

1. This protocol is for the purification of up to 10μg DNA (70bp to 10kb).
2. The yellow color of buffer QG indicates a pH ≤ 7.5. DNA adsorption to the membrane is only efficient at pH ≤ 7.5.
3. Add ethanol (96%-­100%) to Buffer PE before use (see bottle label for volume).
4. Isopropanol (100%) and a heating block or water bath at 50°C are required.
5. All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a convetional table-­top microcentrifuge.
6. Symbols:    ● centrifuge processing; ▲ vacuum processing.

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
2. Weigh the gel slice in a colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100 mg gel ~ 100μl). The maximum amount of gel per spin column is 400mg. For >2% agarose gels, add 6 volumes Buffer QG.
3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every tube every 2-­3 min to help dissolve gel. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10μl 3 M sodium acetate, pH 5.0, and mix. The mixture turns yellow.
4. Add 1 volume isopropanol to the sample and mix.
5. Place a QIAquick spin column in ● a provided 2ml collection tube or into ▲ a vacuum amnifold. To bind DNA, aply the sample to the QIAquick column and ● centrifuge for 1 min or ▲ apply vaccum to the manifold untill all the samples QIAquick column back into the same tube. For example volumes of > 800μl, load and spin/apply vacuum again.
6. If the DNA will subsequently be used for sequenceing, in vitro transcruption, or microinjection, add 500μl Buffer QG to the QIAquick column and ● centrifuge for 1 min or ▲ apply vaccum. ● Discard flow through and place the QIAquick column back into the same tube.
7. To wash, add 750μl Bufick column and fer PE to QIAquickcolumn and ● centrifuge for 1 min or ▲ apply vacuum. ● Discard flow­-through and place the QIAquick column back into the same tube.
8. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
9. To elute DNA, add 50μl Buffer EB (10mM Tris•Cl, pH 8.5) or water to the center of the QIAquick memberane and centrifugethe colum for 1 min. For increased DNA concentration, add 30μl Buffer EB to the center of the QIAquick membrance, let the column stand for 1 min, and then centrifuge for 1 min. After the addition of Buffer EB to the QIAquick membrance, increasing the incubation time to up to 4 min can increase the yield of purified DNA.
10. If the Purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.

Transformation Procedure

Use this procedure to transform One Shot* TOP10 chemically competent E. coli. We recommend including the pUC19 control plasmid DNA supplied with the kit (10 pg/ μl in 5mM Tris-­HCl, 0.5mM EDTA, pH 8) in your transformation experiment to verify the efficiency of the competent cells. Do not use these cells for electroporation.

1. Thaw, on ice, one vial of One Shot® TOP 10 chemically competent cells for each transformation.
2. Add 1 to 5 μl of the DNA (10pg to 100 ng) into a vial of One Shot® cells and mix gently. Do not mix by pipetting up and down. For the pUC19 control, add 10pg (1μl) of DNA into a separate vial of One Shot® cells and mix gently.
3. Incubate the vial(s) on ice for 30 mins.
4. Heat­-shock the cells for 30 secs at 42°C without shaking
5. Remove the vial(s) from the 42°C bath and place them on ice for 2 mins
6. Asceptically add 250 μl of pre-­warmed S.O.C. Medium to each vial.
7. Cap the vial(s) tightly and shake horizontally at 37°C for 1 hour at 225 rpm in shaking incubator.
8. Spread 20-­200 μl from each transformation on a pre-­warmed selective plate and incubate overnight at 37°C. We recommend that you plate two different volumes to ensure that at least one plate will have well-­spaced colonies. For the pUC19 control, dilute the transformation mix 1:10 into LB Medium (e.g. remove 100μl of the transformation mix and add to 900μl of LB Medium) and plate 25-­100μl.
9. Store the remaining transformation mix at +4°C. Additional cells may be plated out the next day, if desired.
10. Invert the selective plate(s) and incubate at 37°C overnight.

Mini and Midi preparation

QIAGEN® Plasmid Mini and Midi Kits

Notes before starting

1. Add RNase A solution to Buffer P1, mix, and store at 2­-8°C
2. Optional: Add LyseBlue® reagent to Buffer P1 at a ratio of 1:1000.
3. Prechill Buffer P3 at 4°C. Check Buffer P2 for SDS precipitation
4. Isopropanol and 70% ethanol are required.
5. Symbols:    ● QIAGEN Plasmid Mini Kit;     ■ QIAGEN Plasmid Midi Kit

1. Harvest overnight bacterial culture by centrifuging at 6000 x g for 15 mins at 4°C.
2. Resuspend the bacterial pellet in ● 0.3ml or ■ 4ml Buffer P1.
3. Add ● 0.3ml or ■ 4ml Buffer P2, mix thoroughly by vigorously inverting 4­-6 times, and incubate at room temperature (15­-25°C) for 5 mins. If using LyseBlue reagent, the solution will turn blue.
4. Add ● 0.3ml or ■ 4 ml prechilled Buffer P3, mix thoroughly by vigorously inverting 4-­6times. Incubate on ice for ● 5 mins or ■ 15 mins. If using LyseBlue reagent, mix the solution until it is colorless.
5. ●: Centrifuge at 14,000-­18,000 x g for 10 mins at 4°C. Re­-centrifuge if the supernatant is not clear
   ■: Centrifuge at ≥20,000 x g for 30 mins at 4°C. Re-­centrifuge the super natant at ≥20,000 x g for 15 mins at 4°C
6. Equlibriate a QIAGEN tip ● 20 or ■ 100 by applying ● 1ml or ■ 4ml Buffer QBT, and allow column to empty by gravity flow.
7. Apply these supernatant from step 5 to the QIAGEN tip and allow it to enter the resin by gravity flow.
8. Was the QIAGEN top with ● 2 x 2 ml or ■ 2 x10 ml Buffer QC. Allow Buffer QC to move through the QIAGEN top by gravity flow.
9. Elute DNA with ● 0.8 ml or ■ 5 ml Buffer QF into a clean ● 2 ml or ■ 15 ml vessel. For constructs larger than 45 kb, prewarming the elution buffer to 65°C may help to increase the yield.
10. Precipitate DNA by adding ● 0.56 ml or ■ 3.5 ml room temperature isopropanol to the eluted DNA and mix. Centrifuge at ≥15,000 x g for 30 mins at 4°C. Carefully decant the supernatant.
11. Wash the DNA pellet with ● 1 ml or ■ 2ml room temperature 70% ethanol and centrifuge at ≥15,000 x g for 10 mins. Carefully decant the supernatant.
12. Air dry pellet for 5­-10 mins and redissolve DNA in a suitable volume of appropriate buffer (e.g., TE buffer, pH 8.0, or 10 mM TrisCl, pH 8.5).

Ligation

Quick Ligation Protocol

1. Combine 50 ng of vector with a 3­-fold molar excess of insert. Adjust volume to 10μl with dH2O.
2. Add 10μl of 2X Quick Ligation Reaction Bugger and mix.
3. Add 1μl of Quick T4 DNA Ligase and mix thoroughly.
4. Centrifuge briefly and incubate at room temperature (25°C) for 5mins.
5. Chill on ice, then transform or store at -­20°C
6. Do not heat inactivate. Heat activation dramatically reduces transformation efficiency.

Enzyme Digestion

PCR

Kits Used

Enzymes

LB Growth Medium

Primers

Gel Electrophoresis