Team:CIDEB-UANL Mexico/WetLab-Protocols

LAB PROTOCOL
IGEM-CIDEB_UANL

General Rules
1. - Use always your lab coat
2. - Pick up always the essential material
3. - Never take any material to your mouth while you’re working with reactants
4. - Keep clean your working place
5. - Every scrap must be in the trash can
6. - Check gas and water keys, depending on your work
7. - Wash your hands before and after every session
8. - Always wear gloves for Ethidium bromide
9. - Save your material after working with it
10. - Every single question, accident, etc. tell to the instructor

Recommendations for Microbiology
11. - Sterilize with heat any crop material before and after using it.
12. - Don’t talk while you are working with sterilized material
13. - When start and finish your, sterilize the surface with 70% alcohol
14. - Al testing tubes, must be vertically
15. - Never deposit a living cultivation in the sump
16. - Sterilize every used material

Laboratory Protocol

Handling of lyophilized DNA’s plates.

This step is for searching and obtaining the desired piece.
Procedure:
1.- Search in the Parts Registry the BioBrick
2.- Drill the plate, and add 10ul of mQ water
3.- Mix very well, and take the liquid to an Eppendorf tube

Preparation for competent cells and their transformation.

This is for preparing the plasmid in the bacterium with thermal shock
Transformation Procedure:
1.- Take 100ul of the bacteria to an Eppendorf tube
2.- Add 2ul of DNA and mix them
3.- Let in ice from 20 to 30 minutes
4.- Dive the Eppendorfs with 42°C water for 1 minute
5.- Take it to ice for 2 minutes
6.- Add LB and incubate them for 20 min, then put them into petri dish at 37°C all the night.

Inoculation in Petri Dish and Test Tube

Inoculate bacteria in dishes with agar and medium tubes with their antibiotics
Procedure:
For planting
1.- Take the colonies in a test tube with LB and the antibiotic
2.- Take the handle sterilized and spread the colonies in the dish.
3.- Incubate for 37°C FOR 16 hours
Reseeding
1.- Add the antibiotic to a test tube
2.- Take with a handle a colony
3.- Then agitate the handle in the Tube, finally incubate at 37°C

Plasmid DNA miniprep

This is for obtaining just the plasmid DNA of a colony
Procedure:
1.- Add 1.5ml of colonies to an Eppendorf, centrifuge 30 seconds and throw the liquid in the trash with soap.
2.- Add 200ul of solution I, II and III in times of 5 minutes respectively and then centrifuge the new solution.
3.- Pass the liquid to and Eppendorf containing 1ml of alcohol at 100%
4.- Incubate at -20°C for 10 minutes
5.- Centrifuge and throw the liquid.
6.- Add 200ul of Ethanol 70% and mix very well
7.- Centrifuge again and throw the liquid
8.- Let the precipitate incubate at 37°C, then add 200ul of mQ water with RNase and mix it
9.- Check in the gel or save it at 4°C

DNA quantification by UV spectrophotometer

This is for quantify the plasmid DNA extracted
Procedure:
1.- Take 1ml of mQ water in an Eppendorf
2.- Add 1ul of plasmid DNA
3.- Prepare the spectrophotometer
4.- Pass the DNA to the spectrophotometer
5.- Select the option (DNA or RNA)
6.- Write the lecture of the spectro at 260, 280 and 320, relation 260/280 and the concentration.

Plasmid DNA characterization

This is for identify the piece we have
Procedure:
1.- Prepare the mix
2.- Deal the mix in equal parts
3.- Add the DNA and mix
4.- Incubate the reactions at 37°C for 1 hour
5.- Use the gel for check the result

BioBrick parts assembly

Procedure:
In this step we are going to cut the plasmid with the appropriate restriction enzymes for deliver the desired fragment and to paste into another bacterium.
1.- Prepare the mix
2.- Lead the mix in equal parts and the add DNA, mix it.
3.- Incubate the reactions for 1 hour
4.- Check in the gel
5.- Save it for its ligation

Ligation of genetic parts

This step is for paste the desired piece in the bacterium we are going to use.
Procedure:
1.- Take in count the concentration of each one of the DNA’s.
2.- Use the calculator for obtain the amounts for preparing the ligation mix at 20ul
3.- Prepare the mix in the next order: mQ water, Buffer, and the plasmid
4.- Lead the mix and add the appropriate ligase
5.- Incubate the reactions at 25°C