Team:CIDEB-UANL Mexico/WetLab-Protocols

General Rules:
1. - Always use your lab coat
2. - Always pick up the used material
3. - Never take any material to your mouth while you’re working with reactants
4. - Keep your working area clean
5. - All non-reusable material must be placed in the correct trash can
6. - Check for oppen gas and water valves, depending on your work
7. - Wash your hands before and after every session
8. - Always wear gloves when working with Ethidium bromide
9. - Every question, accident, etc. tell the instructor

Recommendations for Microbiology
10. - Sterilize with heat any crop material before and after using it.
11. - Don’t talk while you are working with sterilized material
12. - When you start and finish, sterilize the surface with 70% alcohol
13. - Al testing tubes, must be vertical
14. - Never deposit a living cultivation in the sink
15. - 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 for the BioBrick in the Parts Registry
2.- Drill the plate, and add 10ul of mQ water
3.- Mix very well, and take the resusupended DNA to an Eppendorf tube

Preparation for competent cells and their transformation

This i to prepare the plasmid in the bacterium with the 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 a Petri dish.
3.- Incubate for 37°C FOR 16 hours
Reseeding
1.- Add the antibiotic to a test tube previously half filled with Lb medium
2.- With a handle take a colony
3.- Then agitate the handle in the Tube, finally incubate at 37°C

Plasmid DNA miniprep

This is to obtain the DNA plasmid 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