TITLE: pGEM-T Cloning
AUTHORS: Carolina Lopez

[DESCRIPTION]
Protocol for cloning in pGEM-T vector

[BEFORE_START]
PRINCIPLES BEHIND THE PROCEDURE MUST BE UNDERSTOOD. PLEASE CONSULT WITH EXPERIENCED LAB MEMBER THE FIRST TIME YOU USE THIS PROCEDURE. UPDATE AS A GENERAL PROCEDURE AS NECESSARY BUT DO NOT MODIFY WITH SPECIFICS TO YOUR PROJECT, INSTEAD DOWNLOAD AND PASTE A MODIFIED COPY IN YOUR NOTEBOOK. Updated by MH, April 2023

[STEPS]
SECTION: Insert Generation
1. 1. Perform PCR to generate bands of interest in total volume of 50 µL. 
 
 
 
2. Make 1% low melt-agarose gel. 
        a) Mix 1 g of Agar with 100 mL of TAE Buffer. 
        b) Microwave to boil agarose and let cool until you can touch bottle, but gel is not solid.
        c) Add 1.5 µL of EtBr to agarose and pour into DNA gel mold with 10 well comb.
        d) Let gel solidify.
3. Add 10 µL of loading buffer to PCR reaction then Load 60 µL of reaction into well of gel 
4. Run gel for 45 min at 120V. 
5. Visualize band with UV light and cut out section of gel with band with new razor blade and place in 1.5 mL tube. 
6. Purify Band from gel with Promega Wizard SV Gel and PCR Purification Kit (A9282)
        a) https://www.promega.com/products/nucleic-acid-extraction/clean-up-and-concentration/wizard-sv-gel-and-pcr-clean-up-system/?catNum=A9281 
        b) Weigh DNA gel fragment and add 10 µL of Membrane Binding Solution per 10 mg of gel slice. 
        c) Incubate mixture at 65 °C for 10 min or until gel is completely melted. 
        d) Add melted gel mixture to SV minicolumn in Collection Tube and incubate at room temperature for 1 min.
        e) Centrifuge at max speed for 1 min. Discard flowthrough and reinsert column into tube. 
        f) Add 700 µL of Membrane Wash Solution. Centrifuge at max speed for 1 min. Discard flowthrough and reinsert column into tube. 
        g) Add 500 µL of membrane wash solution. Centrifuge at max speed for 1 min. Discard flowthrough and reinsert column into tube. 
        h) Spin empty column for 1 min at max speed to remove excess ethanol. 
        i) Transfer column to labelled 1.5 mL tube and add 35 µL of NF H20. Incubate for 1 min at room temperature. 
        j) Centrifuge at max speed for 1 min. 
        k) Keep eluate and store at -20 °C. 
SECTION: pGEM Ligation Protocol
2. 1. Set up Ligation Reaction in 0.2 mL PCR tube
 
a) *Volume of PCR product should be 2 insert:1 pGEM vector molar ratio
b) For example: 
 

2. Incubate for 60 min at room temperature
3. Transform Product into E. coli
        a) Add 2 µL of product to 50 µL of TOP10 cells and mix up and down slowly with a pipette. 
        b) Incubate for 20 min on ice.
        c) Heat shock bacteria in 42 °C waterbath for 1 min.
        d) Incubate on Ice for 3 min.
        e) Add 100 µL of SOC media and shake in warm room for 60 min.
        f) Plate bacteria onto LB-Antibiotic/Xgal Plate and incubate overnight at 37 °C
                i. Add 40 µL of Xgal to plate and spread with plate spreader. Let dry
                ii. Then plate 100 µL of bacteria onto plate.
                iii. Spread cells with plate spreader to get individual colonies
        g) Pick white colonies for miniprep growth and sequencing.