The iGEM Team of University of Southern Denmark 2012 igem.sdu.2012@gmail.com

mRNA Isolation	PCR	Miniprep	Check Digest
3A-Assembly	Colony-PCR	Transformation	Gel-electrophoresis
Reverse Transcriptase	Mutagenisis	PCR-, Gel Clean-Up	Content

PCR and Gel Clean-Up

Macherey-Nagel NucleoSpin® protocol for PCR clean-up

The following protocol is suitable for PCR clean-up as well as concentration and removal of salts, enzymes, etc. from samples without SDS. Before starting the preparation: Check if Wash Buffer NT3 was prepared according to section 3.
1 Adjust DNA binding condition
Mix 1 volume of sample with 2 volumes of Buffer NTI (e. g., mix 100 μL PCR reaction and 200 μL Buffer NTI).
2 Bind DNA Place a NucleoSpin® Extract II Column into a CollectionTube (2 mL) and load the sample. Centrifuge for 1 min at 11,000 x g. Discard flow-through and place the column back into the collection tube.
3 Wash silica membrane
Add 700 μL Buffer NT3 to the NucleoSpin® Extract II Column. Centrifuge for 1 min at 11,000 x g. Discard flowthrough and place the column back into the collection tube.
Note: Carry-over of chaotropic salt may result in low A260/A230 values. To prevent problems in very sensitive downstream applications or if the entire eluate has to be used, follow the instructions given in section 8.1 (“Suboptimal performance of DNA in sequencing, restriction, or ligation reactions - Carryoverof chaotropic salts”).
4 Dry silica membrane
Centrifuge for 2 min at 11,000 x g to remove Buffer NT3 completely. Make sure the spin column does not come in contact with the flow-through while removing it from the centrifuge and the collection tube.
Note: Residual ethanol from Buffer NT3 might inhibit enzymatic reactions. Total removal of ethanol can be achieved by incubating the columns for 2–5 min at 70 °C prior to elution.
5 Elute DNA
Place the NucleoSpin® Extract II Column into a new 1.5 mL microcentrifuge tube (not provided). Add 15–50 μL Buffer NE and incubate at room temperature (18–25 °C) for 1 min. Centrifuge for 1 min at 11,000 x g.
Note: Yield of larger fragments (> 5–10 kbp) can be increased by using prewarmed elution buffer (70 °C).

Macherey-Nagel NucleoSpin® protocol for DNA extraction from agarose gels

Before starting the preparation: Check if Wash Buffer NT3 was prepared according to section 3.
1 Excise DNA fragment / Solubilize gel slice
Take a clean scalpel to excise the DNA fragment from an agarose gel. Excise gel slice containing the fragment carefully to minimize the gel volume.
Note: Minimize UV exposure time to avoid damaging the DNA.
Determine the weight of the gel slice and transfer it to a clean tube. For each 100 mg of agarose gel add 200 μL Buffer NT.
For gels containing > 2 % agarose, double the volume of Buffer NT. The maximum amount of gel slice per NucleoSpin® Extract II Column is 400 mg or 200 mg of a high percentage gel > 2 %. In this case 2 loading steps are required (step 2).
Incubate sample for 5–10 min at 50 °C. Vortex the sample briefly every 2–3 min until the gel slice is completely dissolved!
2 Bind DNA
Place a NucleoSpin® Extract II Column into a Collection Tube (2 mL) and load the sample. Centrifuge for 1 min at 11,000 x g. Discard flow-through and place the column back into the collection tube.
3 Wash silica membrane
Add 700 μL Buffer NT3 to the NucleoSpin® Extract II Column. Centrifuge for 1 min at 11,000 x g. Discard flowthrough and place the column back into the collection tube.
Note: Carry-over of chaotropic salt may result in low A260/A230 values. To prevent problems in very sensitive downstream applications or if the entire eluate has to be used, follow the instructions given in section 8.1 (“Suboptimal performance of DNA in sequencing, restriction, or ligation reactions – Carryover of chaotropic salts”).
4 Dry silica membrane Centrifuge for 2 min at 11,000 x g to remove Buffer NT3 completely. Make sure the spin column does not come in contact with the flow-through while removing it from the centrifuge and the collection tube.
Note: Residual ethanol from Buffer NT3 might inhibit enzymatic reactions. Total removal of ethanol can be achieved by incubating the columns for 2–5 min at 70 °C prior to elution.
5 Elute DNA
Place the NucleoSpin® Extract II Column into a new 1.5 mL microcentrifuge tube (not provided). Add 15– 50 μL Buffer NE and incubate at room temperature (18–25 °C) for 1 min. Centrifuge for 1 min at 11,000 x g.
Note: Yield of larger fragments (> 5–10 kbp) can be increased by using prewarmed elution buffer (70 °C).