Lab 11
DNA Extraction and PCR
Objective: Students will extract plasmid DNA from E. coli and amplify a 600 bp region of the
genomic FT3 gene using PCR
Extracting plasmid DNA from E. coli with Quiagen Miniprep Kit:
*grow 10 mL liquid bacterial culture overnight in 2XYT w/ 25mg/mL chloramphenicol (should have final OD of
1-1.5)
*spin 10 mL of culture for 15 min at 3500 rpm, resuspend in 250 µL resuspension buffer (P1) containing
.1 mg/mL RNase A
*add 250 µL lysis buffer (P2) and gently invert 4-6 times
*add 500 µL neutralization buffer (N3) and immediately invert gently 4-6 times
*centrifuge the lysate for 10 min at maximum speen in a tabletop centrifuge
*transfer lysates to spin column and spin for 5 min
*wash membrane once with 1 mL buffer PB, twice with 1 mL buffer PE, spinning for 1 min after adding each
buffer
*remove any remaining buffer PE by spinning again for 1 min
*elute DNA by adding 40 µL pre-warmed (70°C) elution buffer (EB) directly to column
*let stand 5 min and spin for 1 min
2XYT:
for 1 L:
16g bacto-tryptone
10g bacto-yeast
extract
5 g NaCl
15 g agar
pH 7 (use 5N NaOH)
PCR:
Each pair of students will prepare one tube of MasterMix, enough for 3 reactions (one for each student
and a negative control).
The two primers to be used are:
FW: FT-ATG – a primer that amplifies at the start codon of both the FT1 and FT3 genes
Sequence: atgtctagcagggagagagaccctc
RVRS: FT-MIDSTOP – an internal primer that amplifies in an intronic region ~600 bp downstream of the
start codon in both FT1 and FT3
Sequence: attccaaggtgatcaatggcacagtg
MasterMix Recipe
Master Mix Recipe
to individual reaction tubes add:
5x green go taq buffer
22 mM MgCl2
5 mM dNTPs
Primer 1 100 ng/µl
Primer 2 100 ng/µl
Taq polymerase
Sterile H2O
per rxn
(µl)
10
5
1
0.5
0.5
0.25
31.75
1:50 dilution plasmid DNA
Total
1
50
per 3 rxns
(µl)
30
15
3
1.5
1.5
0.75
95.25
Cycling Parameters:
95° - 2 min
95° - 30 sec
66° - 30 sec
72° - 45 sec - the extension time is determined by the length of the region being amplified, 1 minute per kb
72° - 5 min
4° - infinite
40 cycles - the number of cycles is the number of times that steps 2-4 are repeated
The PCR products will be saved for next week’s lab.