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transformation

This document outlines a protocol for transforming E. coli bacteria to make them competent for DNA uptake. The steps include growing the bacteria, cooling, centrifuging, resuspending in calcium chloride, adding plasmid DNA, and incubating to allow for recovery and expression of antibiotic resistance. The final step involves plating the transformed bacteria on agar plates to check for colonies after incubation.

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Nani Gopal Saha
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22 views

transformation

This document outlines a protocol for transforming E. coli bacteria to make them competent for DNA uptake. The steps include growing the bacteria, cooling, centrifuging, resuspending in calcium chloride, adding plasmid DNA, and incubating to allow for recovery and expression of antibiotic resistance. The final step involves plating the transformed bacteria on agar plates to check for colonies after incubation.

Uploaded by

Nani Gopal Saha
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd
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1. Pick a Bacterial Colony: Choose a single E.

coli colony from a plate that has been


growing overnight (16–20 hours) at 37°C. Transfer it into 100 ml of SOB (or LB)
medium in a large flask. Grow the culture for 3 hours at 37°C while shaking to ensure
good growth.
2. Cool the Bacteria: Transfer the bacterial culture into ice-cold 50 ml tubes. Keep them on
ice for 10 minutes to cool down.
3. Centrifuge the Cells: Spin the tubes in a centrifuge at 2700g (4100 rpm) for 10 minutes
at 4°C to collect the bacterial cells as a pellet.
4. Remove the Liquid: Carefully pour off the liquid (supernatant) without disturbing the
pellet. Place the tubes upside down on a clean paper towel for about a minute to drain any
remaining liquid.
5. Resuspend in Magnesium-Calcium Solution: Gently mix the bacterial pellet in 30 ml
of ice-cold MgCl₂–CaCl₂ solution by swirling or mild vortexing.
6. Centrifuge Again: Spin the tubes again at 2700g (4100 rpm) for 10 minutes at 4°C to
collect the cells once more.
7. Remove the Liquid Again: Pour off the liquid as before and let the tubes drain for a
minute on a paper towel.
8. Resuspend in Calcium Chloride: Mix the bacterial pellet in 2 ml of ice-cold 0.1 M
CaCl₂ solution (or TFB). This makes the cells competent (able to take up foreign DNA).
o If making the CaCl₂ solution, dilute 10 ml of stock solution in 90
ml of pure water, filter it using a 0.45-µm filter, and chill it to 0°C.
9. Use or Store Cells: At this point, the cells are ready for transformation. Either use them
immediately (Steps 10–16) or store them at -70°C for later use.
10. Add DNA to the Cells: Take 200 µl of the competent cells and put them in a chilled
tube. Add the plasmid DNA (not more than 50 ng) and mix gently. Keep the tubes on ice
for 30 minutes.
11. Heat Shock the Cells: Transfer the tubes to a 42°C water bath for exactly 90 seconds.
This brief heat exposure helps the DNA enter the bacteria.
12. Cool the Cells: Quickly place the tubes back on ice for 1–2 minutes to stabilize the cells.
13. Add Recovery Medium: Pour 800 µl of SOC medium into each tube and incubate at
37°C for 45 minutes. This allows the bacteria to recover and start expressing the
antibiotic resistance gene from the plasmid.
14. Plate the Transformed Bacteria: Take up to 200 µl of the transformed bacteria and
spread them onto agar plates containing antibiotics and 20 mM MgSO₄.
15. Let the Plates Dry: Leave the plates at room temperature until all the liquid is absorbed.
16. Incubate and Check for Colonies: Turn the plates upside down and incubate them at
37°C. Transformed colonies (bacteria that took up the DNA) should appear within 12–16
hours.
This method allows bacteria to take up foreign DNA, usually a plasmid, making them genetically
modified. Let me know if you need further clarification!

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