Transformation

ELECTRO TRANSFORMATION

Transformation may be described as the stable, heritable uptake of exogenous DNA into a host (E.coli) cells that are in a state in which DNA can transform them in their environment are said to be competent. DH10B cells allow efficient cloning of both eukaryotic and prokaryotic genomic DNA. DH10B cells are tested for transformation efficiency using standard protocol. Transformation efficiency should greater than 1.0×10¹⁰ transformants /µg for pUC19 DNA.

In this method transformation done by only applying high voltage ~1.5kv by using Micro Pulser electroporator. Like chemical transformation there is no need of any chemical for proper transformation.

REQUIREMENTS

1. ElectroMAX^TM DH10B^TM cells

          Size :       .5ml

          Store at  -80^oc

2. Ethanol

3. SOB media

Measure        ~900ml distilled water.

Add      :

              20g Bacto Tryptone

              5g yeast extract.

              2ml of 5M Nacl.

               2.5ml of 1M KCl.

              10ml of 1M Mgcl₂

              10ml of 1M Mgso₄ 

-       Adjust the volume to 1L.

-       Autoclave it.

Add   : 20ml of 1M glucose (change to SOC media).

4. Ligation product (10µl)

Required 1µl .

5. BioRad Micro pulser Electroporator.

Adjust to bacterial cells requirement.

6. X-Gal (50mg/ml)
7. IPTG (50mg/ml)
8. Ampicillin (50mg/ml)
9. LB Medium

    For 1000 ml LB ml:

          Tryptone          :     10 g

          Yeast extract    :    5 g

             Nacl                :    10 g

Autoclaved and stored at room temperature.

10.   LB Agar

It consists of LB medium with 15g agar per 1000ml

Autoclaved and stored at room temperature

PROTOCOL

1. Prepare LB/ampicillin/IPTG/X-Gal plates for each ligation reaction. Equilibrates the plate at room temperature.
2. Centrifuge the tubes containing ligation reaction to collect the contents at bottom. Add 1µl of ligation reaction to a sterile 1.5ml MCT on ice.
3. Remove tube of frozen ElectroMAX^TM DH10B cells from storage and place in an ice bucket until just thawed. Mix the cells by gently flicking the tube.
4. Carefully transfer the 20µl of cells into the tube containing 1µl ligation product.
5. Carefully pipette the cell/DNA mixture into a chilled .1cm cuvette. Gently tap the cuvette to ensure that the mixture makes contact all the way across the bottom of the cuvette chamber. Avoid formation of bubbles.
6. Electroporate the sample. Set the optimum condition in electroporator (BioRad Gene Pulser).
7. To the cell in the cuvette, add 1ml of S.O.C medium and transfer the solution to a new MCT.
8. Shake at 200 rpm at 37^oc for 1.5 hour.
9. Plate transformation culture with a volume 50µl,100µl, 200µl onto AXI LB plates.
10. Incubate the plates overnight (16-24 hrs) at 37^oc. If 100μl is plated, approximately 100 colonies per plate are routinely seen using competent cells that are 1 × 108cfu/μg DNA. Use of ultra-high- efficiency competent cells may result in a higher number of background colonies. Longer incubations or storage of plates at 4°C (after 37°C overnight incubation) may be used to facilitate blue color development. White colonies generally contain inserts; however, inserts may also be present in blue colonies.