Team:IIT Delhi/labs

LAB PROTOCOLS

                                                                                                                                                                                                                 

Accurate Experimenting for Accurate Results !!

Transformation

Preparation of competent cells:-

1. Dilute an overnight culture of E. coli 1:200 with LB broth.
2. Incubate at 37°C with shaking (at 200 rpm) until the cells reach early log phase (OD600 = 0.25-0.4).
3. We already have 1X TSS in 4 deg fridge(old). Use it without dilution or thawing. Keep it inside the icebox just after taking out from the fridge.
   OR
   (if the above 1X TSS is not available)
   While cells are growing, thaw 2X TSS on ice and dilute an appropriate amount 1:1 with sterile distilled water (100µl of diluted TSS will be needed for each ml of cells). Chill on ice.
4. Place 2-ml aliquots of early log-phase cells into sterile 2-ml micro-centrifuge tubes and pellet the cells by centrifugation at 4°C at 3000g for 10 min.(6-8 mins for taking part from Igem kit)
5. Remove the supernatant and discard. Add 0.2 ml of the ice-cold 1X TSS and place the tubes on ice.
6. Gently suspend the cells by pipetting.
7. Proceed with the transformation protocol below (Step 2), or immediately freeze cells by immersion in liquid nitrogen or a dry ice/ethanol bath. Store the frozen cells at –70°C.

Transformation:-

1. Thaw frozen TSS-competent cells slowly on ice(if stored at -70°C).
2. Add 100 pg -200 ng (2.5 to 4 ul)(15ul for ligation product)of DNA to each tube of competent cells.
   Note:Addition of more than 10ng of DNA may significantly decrease transformation efficiencies.
3. Flick the tubes to mix the cells and DNA and incubate the cells on ice for 30 minutes.
   
4. Transfer the tubes to water bath/dry bath(42°C) for 90 seconds.
5. Transfer the tubes to ice and incubate for an additional 10 minutes.
6. Add 800 ul (total 1 mL)of LB broth and incubate the cells at 37°C for up to 1 hour with shaking (at 200 rpm).
7. Centrifuge the cells at 3000g for ~ 6min (10 mins after ligation)at 4deg(in temperature control centrifuge).
8. Aspirate the tubes to leave the pellets with 1/4 broth .(keep ~300ul)
9. Plate the cells on-to the appropriate selective or differential medium and incubate overnight at 37°C.Check the procedure for antibiotic.
   1. For Ampicillin: 12ul Amp + 188 ul MQ. In MCT spread it on the culture plate before adding the DNA.
   2. For Chloramphenicol: 1:1000 volume ratio of antibiotic : culture broth. Directly suspend into the culture broth and spread it on the plate.
   3. For Kanamycin: 1:1000 volume ratio of antibiotic : culture broth. Directly suspend into the culture broth and spread it on the plate.
   DNA should be added as soon as the last trace of ice in the tube disappears.
   Incubate on ice for 30 minutes. Expect a 2-fold loss in TE for every 10 minutes you shorten this step.