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From 2010.igem.org

h1. Protocols

Contents 1 Rehydration 1.1 Materials 1.2 Procedure 2 Heat Shock Transformation 2.1 Materials 2.2 Procedure 3 Liquid Culture 3.1 Materials 3.2 Procedure 4 Glycerol Stock 4.1 Materials 4.2 Procedure 5 Miniprep 5.1 Materials 5.2 Procedure 6 Digestion 6.1 Materials 6.2 Procedure 7 Ligation 7.1 Materials 7.2 Procedure 8 Gel Electrophoresis 8.1 Materials 8.2 Procedure 8.3 Making Agarose Gel 8.4 Sample Preparation 8.5 Electrophoresis 8.6 Imaging 9 PCR 9.1 Materials 9.2 Procedure 10 Microinjection

Rehydration

Latest revision: *June 3, 2010*

Materials

• kit plate (from iGEM parts distribution)
• diH ~2~ O

Procedure

1. Using sterile technique, load *10μL* of diH ~2~ O into a p20 pipette.
2. Pierce the foil of the correct well on the distribution plate.
3. Firmly but carefully lower the pipette tip into the very bottom of the well.
4. Mix the DNA with the water by slowly pumping the water in and out of the pipette tip. If done correctly, the water should turn red.
5. Leaving all of the water in the well, stretch parafilm over the plate and then replace its cover.
6. Write "DO NOT TOUCH, KEEP LEVEL" on a piece of tape and affix the tape to the lid of the plate.
7. Ensuring that you keep the plate level, put it into a *4°C* fridge, and leave it there for one hour to allow all of the DNA to dissolve.
8. Transfer the entire DNA solution from the well into an Eppendorf tube.

Heat Shock Transformation

Latest revision: *June 3, 2010*

Materials

• *100μL* of chemical competent cells (TOP10) per transformation
• Miniprep plasmid DNA
• 2XTY or SOC medium
• Antibiotic plates (according to plasmid)

Procedure

1. Take out competent cells from -*80°C* and put them on ice immediately before they are needed.
2. Add *2μl* plasmid DNA to thawed cells and mix by flicking the side of the tube. Incubate on ice for *20* minutes.
3. Pre-warm antibiotic plates in *37°C* incubator.
4. Heat shock for *1* min *15* sec at *42°C*.
5. Place on Ice for *2* minutes.
6. Add *500μl* 2XTY (or SOC) medium (kept at room temp.) to each tube.
7. Shake the tubes at *37°C* for *1* hour on a shaking incubator.
8. Spread *100μl* of each transformation tube on appropriate antibiotic plates.
9. Incubate at *37°C* overnight.

Liquid Culture

Latest revision: *June 3, 2010*

Materials

• Glass or plastic culture tubes
• Growth medium containing appropriate antibiotics
• Glass pipette tubes
• Parafilm

Procedure

1. Flame a glass pipette, open the bottle of medium and flame the mouth.
2. Withdraw amount you need to fill your tubes (*5ml* per tube), flame the cap and recap the bottle as quickly as possible.
3. Remove the tube cap, flame the top of the culture tube, pipette in *5ml*, flame the top of the tube and cap it.
4. Pick up one colony using a P20 pipette tip. Uncap the tube, flame the top, inject the tip into the tube.
5. Incubate the tubes at *37°C* overnight or until cells have reached the desired concentration. This should take between *12* and *16* hours.
6. Seal the transformed bacteria culture plate(s) that were used with Parafilm and store in *4°C* fridge.

Glycerol Stock

Latest revision: *June 3, 2010*

Materials

• Overnight bacterial cell culture
• Cryogenic screw-cap vials
• 30% glycerol in H ~2~ O

Procedure

1. Pipette *750μl* 30% glycerol into cryogenic vials. (Note: withdraw very slowly as glycerol is very viscous)
2. Add *750μl* of overnight culture to each vial.
3. Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed.
4. Label each vial in accordance with labeling convention.
5. Store in a freeze box in a -*80⁰C* freezer.

Miniprep

Latest revision: *June 3, 2010*

Materials

Refer to miniprep kit

Procedure

Refer to miniprep kit

Digestion

Latest revision: *June 3, 2010*

Materials

• Digestion Buffer
• Enzyme(s)
• DNA
• ddH ~2~ O

Procedure

1. Remove the digestion buffer and enzymes from the -*20C* freezer, and place on ice
2. Add *14µL* of water to an Eppendorf tube
3. Thaw the digestion buffer and the enzymes
4. Add digestion buffer to tube
5. Add DNA to tube
6. Add enzymes to tube
7. Incubate (time and temperature are variable and depend upon the enzymes used)

Ligation

Latest revision: *June 3, 2010*

Materials

|Linear vector DNA|*20-100* ng| |Insert DNA|*6:1* molar ratio of insert to vector| |*5X* T4 ligase buffer|*4µl*| |T4 DNA Ligase|*1µl*| |Water, nuclease-free|*15µl* – vector and insert vol | |Total volume|*20µl*|

Procedure

1. Prepare a mastermix that contains T4 ligase buffer and T4 DNA ligase
2. Transfer *5 µl* of the mastermix to each properly labeled sample tube.
3. Add corresponding insert DNA and vector DNA into the tubes. Top the volume to *20* µl using ddH ~2~ O.
4. Incubate one hour at *22⁰C* (or room temperature)
5. Heat inactivate T4 DNA ligase at *65°C* for *10* min.
6. Use up to *5 µl* of the mixture for transformation of chemically competent cells.

Gel Electrophoresis

Latest revision: *June 3, 2010*

Materials

• Loading dye
• 100kb DNA ladder
• 1% agarose gel (*50ml* 1X TBE and *1g* agarose)
• Gel box and power supply
• Ethidium bromide stain
• UV box/gel imager

Procedure

Making Agarose Gel

Weigh out *0.5g* of agarose and add to a *250ml* Erlenmeyer flask.

1. Add *50ml* 1X TBE to the flask and mix by swirling.
2. Microwave TBE agarose until the solution becomes clear and obtains a uniform consistency. (First microwave for *1min* and then for *30sec* intervals. DO NOT allow the solution to boil over in the microwave).
3. Use glove to remove flask from the microwave. Allow flask to cool on the lab bench for *5 min* (But do not wait until the gel starts to polymerize)
4. Take out EtBr from -*20⁰C* freezer. Add *3μl* to *50ml* TBE agarose and swirl the flask to mix. Return EtBr to the freezer immediately after use. (Note: EtBr is a carcinogen and a mutagen. Always use glove and lab coat, if available, to handle things contaminated with EtBr.)
5. Carefully pour TBE agarose into the casting tray to avoid bubbles. Make sure the tray is placed on a flat surface. Insert comb into the TAE agarose gel.
6. Let the gel polymerize for *20min.*

Sample Preparation

1. Add appropriate amount of loading dye such that the mixture of loading dye and sample contains *1X* concentration of dye. If using *5X* dye, use 4 parts sample and 1 part dye.
2. Use appropriate volume of ladder (depends on the ladder used).

Electrophoresis

1. Once the gel is solidified, remove the comb carefully and place the casting tray in the gel box. Make sure the wells point towards the black (negative) electrode.
2. Fill the gel box with TBE until the entire gel is immersed in solution.
3. Load prepared samples into the wells. Slowly pull out the pipette tip from the well before releasing the piston of the pipette. This avoids inserting bubbles into the wells, which will disturb the sample.
4. Close the lid of the gel box. Run the gel at *100V* constant voltage for *1* hour.

Imaging

1. Turn off the gel box power supply.
2. Transport the gel in a plastic box to the dark room. Bring two sets of gloves if you are doing this by yourself, because you cannot touch the computer mouse with EtBr contaminated gloves. The dark room key (with attached USB key) is in the top drawer of the gel box bench.
3. Log in to the computer.
4. Open Genesnap. Click the big green button on the left to take a picture, and manipulate it with the sliders on the right.
5. Print out the gel picture, label each lane, and paste it into your lab book.
6. Save the picture (in .sgd and .jpg formats) in the QGEM folder on the USB stick.
7. Upload the picture to BaseCamp.
8. Remove the gel and dispose of it in the proper waste bin. Wipe down the imaging machine and lock the dark room door.

PCR

Latest revision: *June 3, 2010*

Materials

*10X* PCR buffer (w/ MgCl2)	*5.0* μl
*2* mM dNTP mix	*5.0* μl
*10μM* ddH2O	*33.5* μl
Forward primer (*10μM)	*2.0* μl
Reverse primer (*10μM)	*2.0* μl
Template DNA	*2.0* μl
DNA polymerase (eg. Taq)	*0.5* μl (*1.25unit)
Total volume	*50* μl

Procedure

1. Label PCR tubes with sample names. Always include a negative control sample, which lacks template DNA.
2. Take out reagents from -*20⁰C* freezer and put them on ice at all times, especially DNA polymerase.
3. Make a master mix that contains PCR buffer, dNTP, ddH2O and DNA polymerase. Prepare enough master mix for one more sample than you need in order to compensate for pipetting errors. Return the reagents to the -*20⁰C* freezer.
4. Transfer appropriate volume of master mix into each PCR tube.
5. Add the correct forward and reverse primers to each sample tube.
6. Add template DNA to each tube. (Negative control sample does not have template DNA)
7. Gently vortex the samples or spin down to collect drops.

|Step|Temperature(°C)|Time(min)|Number of cycles| |Initial Denaturation|95|1-3|1| |Denaturation|95|0.5|20-40| |Annealing|T ~m~ -3|0.5|20-40| |Extension|72|1 min/kb|20-40| |Final Extension|72|15|1| |Incubation|10|15|1|

Microinjection