Question

PRELAB ASSIGNMENT - LABORATORY 7 (To be turned in at the beginning of Lab 7) To prepare for this laboratory, read the entire lab and look up any terminology or techniques that you do not understand. 1. Briefly outline (or draw a flow chart for) the procedure that you will use to transform bacteria. Include solutions used, incubation times, and the purpose of each step. 2. What is PGLO? 3. What are the experimental controls that will be used in this laboratory? Explain why these are experimental controls?

Answer 1

1.Bacterial transformation(Dh5alphaor BL21)):

competent cells preparation(chemical)

A) streak fresh culture of bacteria on new lb agar plate and gow overnight at 37C(Fresh culture yiels healthy colonies and reduce contamination)

B)Take single colony from the culture plate and inoculate into 10ml LB media and grow overnight at 37C

C)then use 1ml of overnight culture and inoculate into 100ml LB media and  incubate 2-3 hours at 37C to reach the culture OD 0.4 to 0.6

D)Then pellet down the culture at 4c and add 20 ml of 100mM of CaCl2 .Then incubate it on ice for 20-25 minutes(CaCl2 makes the bacterial cell pores to allow the DNA

E)Then pellet down at 4C and remove the CaCl2  add 1ml of 100mM Cacl2 into pellet ,mix slowly (aliquote 50ul into 1ml eppendorf)

Transformation

1.Take 50 ul of above prepared competent cells and then add 5ul of DNA into it ,vertex properly and incubate it on ice for 20 min

2.Then heatshock at 42C for 70 Seconds and immediately transfer into ice without delay(heatshock takeup the negatively charged DNA present on the lipopolysaccaride molecules on the competent cell surface due to negative potential and cooling will take competent cell inside potential to its normal.This is called the thermal imbalance)

3.incubate it 10 mins on ice and then add 1ml of LB or SOC media into and incubate 1 hour at 37C with shaking(allow the dna for antibiotic resistance)

4.Pellet down the solution and remove the 900 ul of media .

5.then mix the pellet with remaining 100 ul media

6.Then pour the solution on antibiotc resistance LB Agar plate.

7.incubate this at 37C for overnight

2.pGLO: pGLO is a engineered plasmid used to transfer the DNA into bacteria .The 5371(5.371kb) basepairs pGLO plasmid contain gene for green fluorescent protein(GFP),which originally comes from the jellyfish aequorea victoria and expression of this gene is controlled by the promoter from the arabinose operon .In this transcription process sugar used as inducer.This vector also have aminpicillin resistance gene for selecting the bacteria contain plasmid clone from other bacteria

3.Experimental  controls : In the Laboratory experimental controls are used for checking the chemicals and experiments are working or not .Positive and negative controls generally used for the comparision study with your target.