Bacterial Plasmids practical

Chlropehnicol

Ampicillin

Producing isolaeted stock of the plasmids

E.coli plasmids features

• covalently closed

• circular DNA moelcules

• classified into two groups

Two calssified groups

1. e.g sex factor F→

   • exist no more than 5 copies per cell

   • large→ 100kilobases

2. smaller

   • 10 kilobases

   • 20 per cell

Most gene cloning vecotrs use

• second group

• so have higher copy number than natural ancestors

• Some over 100 copies in each cell

Transformation

• Calcium chloride treated cells

• only ‘competent’ cells take up plamid

• then idetified with antiotic resistance (plasmid phenotype)

Plasmid DNA preparation priciples

• DNA molecule is denatured by heating

• hydrogren bonds between complemetanry stads of the double helix broken

• BUT covalent links of DNA backbone remain

• SO

• When linear (or broken circular) becomes denatured

  • two strands completely separate

• BUT PLAMIDS:

  • intertwining of two backbones => strands remain close to one another

  • Once allowed to renature→ stay in solub=ble phase and can be recovered by ethanol precipitation

What happens when linear DNA renatured?

• forms an innsoluble network

• with RNA and proteins

• precipitates

How Agarose gel electrophoresis works

• rate of migrtation through electric field

• proportional to the applied electric field strength

• inversvle prop to size of plasmid

How to transform the E.coli with plasmids

1. incubate L broth with recipient cells

2. chill on ice

3. centrifuge

4. resupend in calcium chlirde

5. centrifuge and add more calcium chloride

6. Add plasmid DNA

7. HEAT SHOCK→ ice bath→ 42 degree heat

8. add L broth

9. incubate→ allows time for newly introduced plasmid genes to be expressed

10. add to plates with Ampiciilin and Chloramphenicol (for the palmid penotpye thing)

Picking and patching

1. Add some colonies of one plate onto the other antibiotic

2. Also use some as a control onto the same antibiotic

3. incubated

4. Notes the phenotpyes

Plasmid Preparation

1. scrap sufficient cells into tube

2. Resuspend in STET buffer

3. add lysis mix with lyzozyme

4. votex

5. 5 mins at room temp

6. 100 degrees dry heat shocking

7. spin

8. through away pellet

9. Add acetate

10. cold isopropanol

11. leave on ice

12. spin and collect pellet

13. dry pellet and dissolve in loading buffer B

Agarose gel electrophoreses

1. run on 0.8% horizontal agraose gel

2. visualised under UV light

Analaysis of gel electrophoresis→ fastest-running band

• monomeric, supercoilded plasmid DNA

• found in vivo

• Can be nicked during stoarage so (probs not very old)

Slower-running

• relaxed circular plasmids

• more common in older plasmid DNA preprartions

or

• Supercoiled dimer

  • formed by recombination between two monomeres

Other bands?

• contaminating chromosomeal DNA

Ways plasmids can exist

1. Supercoiled→ native compressed staet FAST

if gets damaged

2. loses coiled→ open SLOWER

3. linear form→ MORE BANDS

or

4. Forms dimers→ recombination→ x2 on gel but doesn’ affect phenotype