practical 1

Question 1

1st plasmid - pIC19H (non-recombinant, lacZ functional
2nd plasmid - pblt101 (recombinant, contains gene encoding out novel transporter, lacZ disrupted
both plasmid contain ampicillin resistance

Answer 1

practical 1 steps
1. introduce the plasmid into a bacteria and identify bacteria containing the plasmid
2. purify the plasmid plC19H from bacteria (miniprep)
3. test the purified plasmid pblt101 to confirm it contains the insert and determine the orientation of the insert (PCR)
4. restriction digestion of recombinant plasmid
5. analysis of recombinant DNA and PCR products by agarose gel electrophoresis

Question 2

1. transformation and selection

Answer 2

make competent E.coli
transformation - plasmid introduces to E.coli by heat shock
selection - only E.coli containing the plasmid will survive in the presense of the antibiotic ampicillin
problem - ampicillin selection only tests for the presence of the plasmid, not whether the insert is in the plasmid (use blue/white selection)
blue and white selection - if the insert is in the plasmid it will disrupt the lacz gene as thats were the plasmid is cut and where the gene is ligated, disrupted lacz causes white colonies, undisrupted lacz causes blue colonies (no gene insertion)

Question 3

2. plasmid DNA purification (miniprep)

Answer 3

commercial kit
extract plasmid out the cultured bacteria
have previously selected a single colony of bacteria that contained the plasmid
grew bacteria culture overnight
remove the plasmid from the bacteria
follow steps

Question 4

3. PCR

Answer 4

ds DNA template
95°C - denature
50-65°C - primer annealing
72°C - taq DNA polymerase
repeat steps

Question 5

IPTG

Answer 5

When IPTG is added, the repressor lifts off the operator. This allows more RNA polymerase to bind, increasing the transcription (and later translation) of LacZ into β-gal.