transformation and preparation of competent cells

8/10/2019 Transformation and Preparation of Competent Cells

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MATERIALS REQUIRED:

Glassware : Culture Tubes, Conical Flasks, Beakers, Measuring Cylinders, Spreader, Petri-

plates

Apparatus: Shaker Incubator, Spectrophotometer, Laminar, Centrifuge, Water bath,

Micropipettes and Microtips, 1.5 ml Microfuge tubes, Polypropylene Oakridge tubes,

Crushed Ice, Blotting Paper, Toothpicks or Inoculation Loop

Chemicals Required:

CHEMICAL STOCK CONC. WORKING CONC. MOL. WT.

CaCl2 1 M (50ml) 100mM and 20mM 111 g/l

MgCl2 1 M (50ml) 80mM 203.31 g/l

Tris-Cl 1 M (250ml) 121 g/l

EDTA 0.5 M (100ml) 186 g/l

Ampicillin 100 mg/ml (1000X) 1 X ?

Carbenicillin 50 mg/ml (1000X) 1 X ?

Tetracyclin 50 mg/ml (1000X) 1 X ?

Glycerol 100% 15%

TE BUFFER: (For 100 ml):o 1 ml of 1 M Tris-Clo 20ul of 0.5 M EDTA

SOC MEDIA: ( For 100ml):o 2.0g Tryptoneo 0.5g Yeast Extracto 0.5g NaClo 0.36g Glucose

100 ml of MgCl 2 (80mM) - CaCl 2 (20mM) solution 300 ml LB Agar (For 8+1 plates) 100 ml for LB Broth + 10 ml LB Broth in a culture tube

Culture used: E. coli strain DH5 .

DNA Plasmid: pBR322 (Stock Concentration: 330ng/ )

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Plasmid Dilution: Working Concentration: 1 ng/ ul.

This can be achieved by serial dilution.

330 ng/ul 10 ng/ul 1 ng/ulTRANSFORMATION EFFICIENCY: (CFU/g plasmid)

Number of colonies x 1000

% cells plated x Amount of DNA added (ng)

EXPERIMENTAL SET UP:

In order to check the success of transformation, you will maintain 2 sets of plates, TEST and

CONTROL.

PLATING SCHEME FOR TEST: (% Check up)

1000l E.coli + pBR322

LB only ( ) LB + Amp +Tet ( )

LB+Amp ( ) LB+Amp+Tet ( )

LB+Tet ( )

PLATING SCHEME FOR CONTROL:

1000l E.coli + TE Buffer

LB only ( ) LB+Tet ( )

LB+Amp ( )

PREPARATORY STEPS:

1. All working concentrations of reagents to be used for this experiment must be prepared from the Stock Solutions.

2. As per the plating scheme illustrated above, prepare LB Agar required for pouring 8

plates. Also account for 1 plate which you will use to subculture E. coli for this

experiment.

3. Autoclave all glassware, reagents, LB Broth and LB Agar.

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PREPARATION OF COMPETENT CELLS

1. Pick a single , fresh bacterial colony (2-3 mm) in diameter from the subcultured E.coli

DH5 plate that has been incubated for 16-20 hours at 37 C. Transfer the colony into

100ml of LB Broth in a 500ml flask. Alternatively, you may use 50-100 l of log-

phase starter culture for inoculation into broth. Incubate the culture for 3 hours at

37C in a shaker incubator at 200 RPM.

For efficient transformation, it is essential that the number of viable cells not exceed

108 which corresponds to an OD of 0.4. (Ensure that the OD DOES NOT cross this

value)

2. Transfer the bacterial cells to sterile, disposable, ice-cold 50ml polypropylene tubes or

Oakridge tubes. Cool the cultures to 0 C by storing the tubes on ice for 10 mins.

3. Recover the cells by centrifuging at 2700g for 10 min at 4 C .

You should observe a small pellet

The centrifuge should be prechilled to 4 C before use.

4. Decant the medium from the cell pellets. Stand the tubes in the inverted position on

blotting paper for 1 min to allow the last traces of media to drain away.

It is preferred that this step be done in the laminar to avoid contamination from Amp R

Bacteria in the surroundings

5. Resuspend each pellet by swirling or gentle vortexing in 30ml of ice-cold MgCl 2-

CaCl 2 solution (80mM MgCl 2, 20mM CaCl 2)

You will observe that the pellet becomes fluffy

6. Recover the cells by centrifugation at 2700g for 10 mins at 4 C

7. Decant the medium from the cell pellets. Stand the tubes in the inverted position on

blotting paper for 1 min to allow the last traces of media to drain away.

8. Resuspend the pellet by swirling or gentle vortexing in 2 ml of ice-cold 0.1 M CaCl 2

for each 50 ml of original culture.

9. At this point, you have 2 options

a. Use the cells directly for transformation

b. Dispense into aliquots of 200l as 15 % Glycerol Stock in prechilled 1.5 ml

eppendorfs using pre-chilled tips and freeze at -70 C.

Comment [R4]: Sir, I haven t mentioned theculturing of DH5 in culture tubes from which wedo streak plating. Is it required? Directly startedpick from plate .


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TRANSFORMATION

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8. Include all positive and negative controls. In this experiment, you are transforming

competent E.coli with pBR322 which has Amp R and Tet R . Please refer to the

EXPERIMENTAL SETUP for details on the plating scheme.

9. Take 8 tubes each containing aliquots of 200ul of competent cellsprepared and add

the plasmid DNA (No more than 50ng in a volume of 10l or less) to the Test tubes

only.To the Control tubesa dd an equal amount of TE buffer instead. Mix the

contents of the tubes by swirling gently. Store the tubes on ice for 30 mins.

10. Transfer the tubes to a rack placed in a preheated 42 C circulating water bath. Store

the tubes in the rack for exactly 90 seconds. Do not shake the tubes.

Heat shock is a crucial step. It is very important that the cells be raised to exactly the

right temperature at the correct rate.

11. Rapidly transfer the tubes to an ice-bath. Allow the cells to chill for 1-2 mins.

12. Add 800l of SOC M edium to each tube (thus making the contents of each tube 1000

l). Incubate the cultures for 45 mins in a water bath set at 37C to allow the bacteria

to recover and to express the antibiotic resistance marker encoded by pBR322.

13. Transfer the appropriate volume of transformed competent cells using spread plate

technique onto the LB Agar plates which have the appropriate antibiotic.

To plate rest, spin down the remaining contents of the tube, discard the supernatant and

plate the now concentrated remainder.

14. Invert the plates and incubate at 37 C. Transformed colonies should appear in 12-16

hours. Store the plates at 4C after colonies appear.

EXPECTED RESULTS:

LB ONLY LB + AMP LB+ TET LB+AMP+TET

WITH pBR322 + + + +





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transformation and preparation of competent cells

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