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From 2010.igem.org

MiniPrep

Alkaline Lysis

Alkaline Lysis is for screening of plasmids

1. Pellet the overnight culture(s) in a 1.5 ml or 2ml eppendorf tube. (I usually do 10,000 rpm, 3 minutes) 1 minute works fine. I usually use 3 ml culture per prep.
2. Resuspend each pellet in 200 μl Alkaline Lysis Sol I, RnaseA added (final RNase A concentration should be 100 μg/mL). Make sure there are no lumps.
3. Add 400 μl Alkaline Lysis Sol II. Invert 4-6 times to mix. Do not allow reaction to lyse for more than 5 min. Sample should clarify.
4. Add 300 μl Alkaline Lysis Sol III. Invert 4-6 times to mix. Sample should have a white precipitate.
5. Add 100 μl chloroform. Do this in a fume hood. Invert 4-6 times to mix.
6. Rest on ice for 5-10 minutes. This step is so that the chloroform does not get too hot in the centrifuges and leak out of the tubes. If you want to skip this step you might consider using less chloroform. I put the tubes at -20 for a couple of minutes.
7. Centrifuge at max. speed (14,000 rpm) for 10 minutes.
8. Pipet 750μl of supernatant/aqueous layer into a fresh tube. I do up to 800 uL
9. Add 1/10 volume (75μl) 3M NaOAc, pH 5.2. Vortex/flick to mix. 80 uL
10. Add 0.7-1.0 Volume COLD isopropanol. Vortex/flick to mix. If in a hurry go straight to step 11, otherwise rest on ice for 10-30 minutes. I have even let it precipitate overnight at 4°C if convenient. 600 uL isopropanol. Then I put it at -20 for 5 minutes up to over the weekend if needed.
11. Centrifuge at max. speed for 25 min. Most miniprep protocols say to do this at 4°C, but I have not noticed decreased yield by centrifuging at room temp.
12. Remove and discard the supernatant. Don’t disturb the pellet. Sometimes I can’t see a pellet, and more often than not I still have DNA.
13. Add 1ml of 70% EtOH (at room temp.). Invert 4-6 times to rinse the tube.
14. Centrifuge at max speed for ~5 minutes. Room temp. is fine. Remove and discard the EtOH.
15. Repeat steps 14 and 15 to remove all traces of isopropanol. Pulse spin after removing bulk of final EtOH wash and pipet off remaining EtOH.
16. Air dry the pellet for ~15 minutes (pellet will change from white to clear as it dries). Resuspend in desired volume of H2O or T10E1, depending on downstream applications. If you pipet off the EtOH well, then I have done this for as little as 2 minutes before. For fosmids, I usually resuspend the pellet in 20 uL water.

Kit

Use the kit when you need very clean DNA. Ex cloning, sequencing

1. Refer to kit instructions

Digestion

Screening

For screening, you only need a small amount to run on a gel - 10uL rxn

Check enzyme compatibility, what buffer is needed, and whether BSA is necessary

• DNA - 2uL (usually fine)
• Buffer(10x) - 1uL
• BSA(10x) - 1uL
• Enzyme - 0.4uL each (ADD LAST and no more than 10% of rxn volume)
• Water - fill to 10uL

1-2 hours in 37C waterbath (check NEB if you want quicker time)

• Add 2uL of 6x Dye
• Load 6uL in gel

Cloning

During cloning, you will need to digest more DNA for gel extraction - 50uL rxn

Check enzyme compatibility, what buffer is needed, and whether BSA is necessary

• DNA - 2ug
• Buffer(10x) - 5uL
• BSA(100x) - 0.5uL
• Enzyme - 2uL each (ADD LAST and no more than 10% of rxn volume)
• Water - fill to 50uL

1-2 hours in 37C waterbath (check NEB if you want quicker time)

• Add 10uL of 6x Dye
• Load 60uL in gel

Template Destruction

If your product for digestion came directly from PCR you can destroy the original template by preforming a DpnI digestion. DpnI will digest methylated DNA. PCR product is unmethylated. If needed, do this step before cloning digestion.

• 1uL DpnI/50uL rxn
• incubate in 37C waterbath for 1 hour

Gel Extraction

• "Excise the DNA fragment from the agarose gel with a clean, sharp scalpel."

Minimize the size of the gel slice by removing extra agarose."

• "Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 μl).

For example, add 300 μl of Buffer QG to each 100 mg of gel. For >2% agarose gels, add 6 volumes of Buffer QG. The maximum amount of gel slice per QIAquick column is 400 mg; for gel slices >400 mg use more than one QIAquick column.

• "Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2–3 min during the incubation."

IMPORTANT: Solubilize agarose completely. For >2% gels, increase incubation time.

• "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 10 μl of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow. The adsorption of DNA to the QIAquick membrane is efficient only at pH ≤7.5. Buffer QG contains a pH indicator which is yellow at pH ≤7.5 and orange or violet at higher pH, allowing easy determination of the optimal pH for DNA binding.

• "Add 1 gel volume of isopropanol to the sample and mix."

For example, if the agarose gel slice is 100 mg, add 100 μl isopropanol. This step increases the yield of DNA fragments <500 bp and >4 kb. For DNA fragments between 500 bp and 4 kb, addition of isopropanol has no effect on yield. Do not centrifuge the sample at this stage.

Ligation

Transformation

Plating

PCR

PCR clean-up

Colony PCR

Electro-competent Cells

Freeze Stock

Making Primers

Diluting Primers

Using the Autoclave