RFLP Screen to Distinguish Between Recombinants
Wong
After generating a library of 16S rDNA clones, clones can be screened for duplicates by using a
combined PCR-restriction enzyme RFLP technique. Each clone is colony-amplified in a PCR
reaction. Then restriction enzymes (multiple 4-cutters) are added to generate a restriction
pattern unique to that clone. When these digests are displayed on an agarose gel, it becomes
easy to distinguish clones from one another, and duplicates are eliminated from further
consideration.
Set up 5 µL PCR reactions.
Thaw solutions quickly (hand warmth), and vortex briefly. Prepare master mix under PCR-clean
conditions (sterile hood with gloves and sleeve protectors), with all reagents kept cold (on ice
or cold box)
PCR MASTER MIX (stock reagents from Qiagen)
Example of 10 rxns
10x PCR Buffer
5
BSA (1 mg/mL non-acetylated stock; Sigma)
4
dNTP mix (10 mM each dNTP)
1
63F/1387R primer mix (5 pmol/µL each in mix)
4
Taq DNA polymerase
0.25
H2O
35.75
________
Total Master Mix volume
50 µL
Add Master Mix to 200 µL PCR tubes kept on cold rack.
Each Sample
Master Mix
10 µL
Touch sterile micropipet tip into colony or culture, and transfer some cells to Master Mix.
(If using a colony, also transfer some cells to a 0.5 mL tube of sterile LB broth supplemented
with Ampicillin [50 µg/mL final conc], and shake at 37°C, 1-2 days, to generate a culture for
plasmid isolation).
PCR CONDITIONS
Start up thermocycler ahead of time to allow lid to prewarm.
Bio-Rad iCycler or MyCycler thermocycler
95°C, 5 min preheat; 35 cycles of 94°C, 30 sec / 55°C, 30 sec / 72°C, 2 min; 72°C, 7 min final
extension; 4°C hold.
Run usually takes about 3 hours to complete.
Freeze samples until next step, or place on freezer blocks if processing immediately.
RFLP Screen to Distinguish Between Recombinants
Wong
RESTRICTION ENZYME DIGESTION
PCR reactions are supplemented with restriction enzyme buffer and enzymes to generate a
restriction fragment pattern. Ideal restriction enzymes would be those with a short DNA
recognition sequence (e.g. “4-cutters”, such as HhaI [GCGC] and RsaI [GTAC]) that would be
likely to cut frequently, and generate restriction fragment length polymorphism (RFLP) patterns
that would distinguish between similar sequences.
Set up a restriction digestion master mix to add 10 µL to each PCR reaction.
RESTRICTION MASTER MIX
Example of 10 rxns
H2O
88
10x Buffer C (Promega)
10
HhaI (Promega; 10 Units/µL stock)
1
RsaI (Promega; 10 Units/µL stock)
1
________
Total Master Mix volume
100 µL
Add 10 µL Restriction Master Mix to each PCR reaction.
37°C incubation for minimum 3 hours.
ASSESSING RFLP PRODUCTS
For gel analysis of RFLP products, prepare a 1.5 % agarose gel, 0.5x Lithium Borate (LB)
running buffer. Ethidium Bromide can be included in the gel or used post-run to stain.
Add 1.5 µL 10x Loading Dyes to each sample. Load gel.
Load 5µL BioLine Easy Ladder I as size markers.
Run gel at 300 V until Orange G dye front is at least 6 cm from wells.
View gel on UV transilluminator or Dark Reader Blue transilluminator.
Photograph using a dark orange filter.
SELECTING RELEVANT CLONES
Eliminate one member of any pair of clones that have the same RFLP pattern. From the rest of
the clones, grow up cultures and isolate plasmid DNA (see MoBio UltraClean Mini Plasmid Prep
kit).