IGEM BOOT CAMP SUMMER 2014

LAB PROTOCOL PREPRATION OF COMPETENT CELL (E.coli DH5 alpha strain) INTRODUCTION OF PLASMID INTO THE COMPETENT CELL PREPARING COLONIES OF TRANSFORMED CELL

PRE-PREPRATION Autoclaved : 80mm MgCl mm CaCl2 sol. Autoclaved: 100mm CaCl2 sol. Autoclaved : 100 ml LB(Luria Bertani Broth : 2.5gm in 100 ml distilled water) Autoclaved : 10 ml LB(Luria Bertani Broth : 0.25gm in 10 ml distilled water) Autoclaved:various lab apparatus such as beaker,conical flask,test tube etc. Alcohol solution (70% v/v)

PROTOCOL (PREPARING OF COMPETENT CELL) Inoculation of 10 ml LB liquid media with (40 ul Ecoli ) and left to incubate overnight. From overnight grown culture take 1% inoculum to inoculate into larger LB media(100ml) contained in a 250 ml conical flask. The larger flask was put in the incubator at 37 degree so as to obtain absorbance of ( ) at wavelength of 600nm(it takes around 2-3 hours). Absorbance was calculated through a sophisticated device known as Spectrophotometer,absorbance was calculated wrt to pure LB media and was observed around ( 0.48) in our case. The cultured Ecoli was put into ice for half an hour and then into centrifuge at 3300 rpm for 5 minutes to separate the Ecoli in the form of pellet after removing supernatant obtained.

PROTOCOL (PREPARING OF COMPETENT CELL) The pellet obtained was transferred into the MgCl2 + CaCl2 sol prepared previously and dissolved(for about 1 hour).And the dissolved mixture was put into centrifuge for 5 minutes at 3300 rpm. Supernatant obtained was separated from the pellet and pellet was transferred to 2ml of CaCl2 sol (preprepared) and kept in refrigerator at 4 degrees in a beaker filled with ice for the night.

PRE-PREPRATION Autoclaved: LB agar plates which consist of (LB broth + agar gel + Xgal + IPTG)

PRE-PREPRATION

PROTOCOL (INTRODUCTION OF PLASMID INTO THE COMPETENT CELL) Inoculation of 100ngm of DNA(puc18) into 200ml of competent cell. It was placed on ice for minutes and then put for waterbath at 42 degrees(heat shock) for about seconds. Snapfreeze : It was transferred to ice for 5 minutes. Addition of 1 ml LB broth into the solution and then put to incubate at 37 degrees in incubator for around minutes.

PROTOCOL PREPARING COLONIES OF TRANSFORMED CELL The prepared solution was put into centrifuge at 5000 rpm for 30 seconds and the supernatant was removed from it leaving 100 ul of transformed ecoli bacteria Rest of the work was done under lamina hood. All the equipment were sterlized with alcohol solution and the 100 ul of prepared transformed cell were transferred to the petridish containing LB agar media and spread with a spreader and then left to get cultured under proper environment i.e in incubator at 37 degree.

EXPECTED RESULT

RESULT

In one of the petridish there was no colonies which can be seen with naked eyes expressing blue colour. In the other there was just one colony of transformed ecoli bacteria depicting blue colour The ecoli formed colonies in a form of lawn as the plasmid we expressed did not had gene which expresses ampicillin resistant nature in them so even non transformed ecoli bacteria were also cultured. Fungal infection was found on the plates

SECOND ATTEMPT

PRE-PREPARED Autoclaved : 80mm MgCl mm CaCl2 sol. Autoclaved: 100mm CaCl2 sol. Autoclaved : 100 ml LB(Luria Bertani Broth : 2.5gm in 100 ml distilled water) Autoclaved : 10 ml LB(Luria Bertani Broth : 0.25gm in 10 ml distilled water) Autoclaved:various lab apparatus such as beaker,conical flask,test tube etc. Alcohol solution (70% v/v)

CORE STEPS PREPRATION OF COMPETENT CELL (E.coli DH5 alpha strain) INTRODUCTION OF PLASMID INTO THE COMPETENT CELL PREPARING COLONIES OF TRANSFORMED CELL

MAIN DIFFERENCE All the steps taken to complete it were same,the only difference this time was that we had pre-prepared culture,practice so it was completed in 2 days only 1 st day : prepared competent cell (the solution containing ecoli + CaCl2 was left overnight) 2 nd day: spreading of the solution on LB agar plates and left overnight.

RESULT No Colonies formed expressed blue colour in both the agar plates Fungal infection

CONFUSED

WHAT TO DO NEXT?

THIRD ATTEMPT This time the experiment was not conducted by us but by our ******** It was done just to carefully notice our mistake by observing the way it was done by *******

POSSIBLE ERRORS The LB agar plates containing Xgal and IPTGmight have been not prepared properly. LB(Luria Bertani) used was of not of good quality(in terms of nutrient content) Proper cleaniness and lab ethics were not seriously followed. Transformation efficiency might have been decreased due to rough handling of cells during the preparation of competent cell.

OTHER PROGRESS

READINGS: Previous year iGEM ProjectsTEAM 1 : Notebook completed till may,about project. TEAM 2 : Introduction to bio synthetic,dow agrosciences,about project,notebook has to be completed in a proper before giving presentation.

FUTURE PLANNING Further reading about various techniques like PCR, Gibson assembly and practically doing them.And reading of other projects.

ACKNOWLEDGEMENT SAHARS KUMAR : mentor during the entire summers guided us all along the iGEM boot camp. BHUPENDRA SIR : helped us along the experiment regarding lab protocol,lab ethics. AARUSHI MAM: guided us during the lab work. THANKS A LOT

iGEM BOOT CAMP SUMMER 2014