Team:Wisconsin-Madison/protocols

From 2010.igem.org

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==Transformation==
==Transformation==
 +
*Clean the cuvette and UV for 15mins.
 +
*Locate Electroporator source and cuvette holder.
 +
*Thaw required number of frozen cell aliquots on ice
 +
*Thaw required DNA on ice
 +
*put the clean cuvette on ice
 +
*mix 0.5ul to 1.0ul DNA with 40ul competent cells
 +
*let it sit on ice for 2-5mins.
 +
*transfer the DNA-Cell mixture into the cuvette
 +
*Place the cuvette in the holder
 +
*Have 960ml of LB broth ready
 +
*press the button to "shock" the cells
 +
*immediately put 960ml LB into the cuvette and mix well
 +
*transfer the cells into a 1.5ml centrifuge tube
 +
*outgrow transformed cells in 37C shaker or water bath for 1-1.5hour.
 +
==Plating==
==Plating==
==PCR==
==PCR==

Revision as of 06:10, 30 September 2010

Contents

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.

  • Place a QIAquick spin column in a provided 2 ml collection tube.
  • To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min.

The maximum volume of the column reservoir is 800 μl. For sample volumes of more than 800 μl, simply load and spin again.

  • Discard flow-through and place QIAquick column back in the same collection tube.

Collection tubes are re-used to reduce plastic waste.

  • To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min.

Note: If the DNA will be used for salt sensitive applications, such as blunt-end ligation and direct sequencing, let the column stand 2–5 min after addition of Buffer PE, before centrifuging.

  • Discard the flow-through and centrifuge the QIAquick column for an additional 1 min at ≥10,000 x g (~13,000 rpm).

IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.

  • Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
  • To elute DNA, add 50 μl of Buffer EB (10 mM Tris·Cl, pH 8.5) or H2O to the center of the QIAquick membrane and centrifuge the column for 1 min at maximum speed. Alter- natively, for increased DNA concentration, add 30 μl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min.

IMPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. The average eluate volume is 48 μl from 50 μl elution buffer volume, and 28 μl from 30 μl. Elution efficiency is dependent on pH. The maximum elution efficiency is achieved between pH 7.0 and 8.5. When using water, make sure that the pH value is within this range, and store DNA at –20°C as DNA may degrade in the absence of a buffering agent. The purified DNA can also be eluted in TE (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.

Ligation

  • measure the concentration of the inserts and the vectors.
  • use In-Fusion® Molar Ratio Calculator from Clontech to calculate the mixing ratio of the inserts and vectors

use Insert/Vector Ratio: 3-5

  • once the amount of inserts and vectors has been calculated, make a calculation for a 10ul total volume reaction
  • After the calculation has been done, get a small tube and label it
  • place the T4-buffer on ice to let it dissolve
  • Add the insert and vector into the tube, mix (Add water to compensate if needed)
  • Add 1ul T4-buffer into the mixing solution
  • Add 1ul Ligase into the mixing solution
  • Either leave it on the bench for a bench-top ligation for 2 hours

OR put into the thermocycle at 16C for overnight

Transformation

  • Clean the cuvette and UV for 15mins.
  • Locate Electroporator source and cuvette holder.
  • Thaw required number of frozen cell aliquots on ice
  • Thaw required DNA on ice
  • put the clean cuvette on ice
  • mix 0.5ul to 1.0ul DNA with 40ul competent cells
  • let it sit on ice for 2-5mins.
  • transfer the DNA-Cell mixture into the cuvette
  • Place the cuvette in the holder
  • Have 960ml of LB broth ready
  • press the button to "shock" the cells
  • immediately put 960ml LB into the cuvette and mix well
  • transfer the cells into a 1.5ml centrifuge tube
  • outgrow transformed cells in 37C shaker or water bath for 1-1.5hour.

Plating

PCR

PCR clean-up

Colony PCR

Electro-competent Cells

Freeze Stock

Making Primers

Diluting Primers

Using the Autoclave

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