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Preparation Of Lb Broth And LB Agar Preparation

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3.0 Material and Methods 3.1 LB Broth and LB Agar Preparation LB Broth powder and LB Agar powder (Sigma-Aldrich) were used for preparation of LB Broth and LB Agar respectively. For LB Broth, 35 g of LB Broth powder was added into 1 L of ddH2O. The mixture was heated up to boiling until all the LB Broth powder was dissolved. For LB Agar, 25 g of LB Agar powder was added into 1 L of ddH2O. The mixture was then allowed to stir to suspend. Both LB Broth and LB agar mixture were allowed for autoclave for 15 minutes at 121 °C to sterilize. Both solutions were allowed to cool slightly before making additions, such as antibiotics (ampicillin). LB agar was prepared by pouring LB agar solution into petri dishes and allowed to solidify. The petri dishes were seal with parafilm tape and inverted before store at 4 °C together with LB Broth. 3.2 Colony Screening from Glycerol Stock Petri dishes contain LB Agar that was prepared was used for streak plate method. Next, flame the inoculum loop and wait for it to cool down. The inoculum loop takes a drop of the liquid culture medium (Glycerol Stock) and spread carefully in a line across the surface of the agar. Sterilise the loop in the flame again and allow it to cool. Turn the petri dishes so the previous line can be the start of the next ones. After repeated several times, close the petri dish immediately and inverted before seal with parafilm tape. The LB Agar was left for growth in the incubator overnight (12-16 hours) at 37 °C which can be used as DNA template for Colony PCR. 3.3 Colony PCR Single colonies were selected using sterile 10 μl pipette tip and inoculated with 0.2 PCR tube. Colony PCR was performed with each reactions consists of around 1 μl cell culture, 2.5 pmol of Grb14 forwa... ... middle of paper ... ...rm (1:1) was added to the linearized sample in a 1.5 ml microcentrifuged tube. The mixtures were centrifuged at 13000 rpm for 2 minutes at room temperature. The upper aqueous solution was transferred to another sterile 1.5 ml microcentrifuged tube. Equal volume of chloroform was added and centrifuged at 13000 rpm for 5 minutes. Again, the aqueous solution was transferred to a new 1.5 ml microcentrifuged tube and 1/10 volume of 3M Sodium Acetate Solution was added. Then, 2.5 volumes of cold absoluted ethanol were added to precipitate the DNA. The mixture was incubated in -20 °C for overnight or -80 °C for 1-2 hour. After incubation, the mixture was then spinning at 13000 rpm for 30 minutes at 4 °C. The supernatant was discarded and wash with 70% cold ethanol. The pellet was allowed to air dry for 20-30 minutes and resuspend in water, nucleus free water or TE buffer.
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