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Not everything that can be counted counts,
and not everything that counts can be counted.
Albert Einstein
Day 3: DNA verification
Overview of Experiment
In today's lab, you set up several types of PCR reactions for
the colony screen: the negative control demonstrates
that in the absence of a specific template DNA, the primers
alone do not amplify a specific product; the reactions using
plasmid DNA are for amplification of specific BioBricks; colony
PCR is used to screen putative recombinants after
ligation/transformation. Additionally,
you evaluate the transformations--what evidence, if any, do you have
that your genetic circuit is working?
Background
PCR Colony Screen
PCR of an individual bacterial colony is a quick and relatively
easy method to screen transformants. We use forward and reverse
primers that bind upstream and downstream, respectively, of
the multiple cloning site (MCS) on BioBricks or an internal
primer (i.e., one that anneals to the insert DNA) with one of
the MCS primers. The size of the product generated varies with
the insert present in the BioBrick. Thus, any single colony producing
an amplified fragment of the expected size is likely to contain
the desired plasmid DNA. We use
OneTaq® Hot
Start Quick-Load® 2X Master Mix with Standard Buffer to
screen colonies resulting from ligation of BBa_R0040 and BBa_E0840.
Because we are using a "hot start" mix, reactions can be set
up at room temperature. After the PCR run is complete, you analyze the PCR reactions for the colony
screen on an agarose gel.
PCR Reactions:
- Preparation of PCR reactions in the order given in Table
1 minimizes contamination of the stock solutions and the
samples
- A 0.2 or 0.5 ml tube size is required to fit into the thermal
cycler.
Note: Label the tubes on the lids.
- Each column in the table represents a single tube
- Reactions will be performed in 50 µl final volumes
- You will have a TOTAL of 6 or 7 PCR reactions: 1 negative
control; 2 using plasmid DNA (diluted 1:100 in nuclease-free
water); 3 or 4 with individual bacterial colonies
Table 1: Construction of PCR Reactions. Add
reagents in the order listed here.
PCR Reactions: |
Negative Control (no DNA) |
Plasmid DNA (X2)
|
Bacterial
Colony (X4) |
2X Master Mix |
25 µl |
25 µl |
25 µl |
Nuclease-Free
Water
(NF H2O) |
19 µl
|
19 µl
|
20 µl
|
Forward Primer (1 µM final concentration) |
2.5 µl
VF2
|
2.5 µl
VF2
|
2.5 µl
VF2
|
Reverse Primer (1 µM final concentration) |
2.5 µl
VR
|
2.5 µl
VR
|
2.5 µl
VR
|
DNA Template |
1 µl nuclease-free water
|
1 µl
plasmid DNA (P) (1:100)
|
"touch"
bacterial colony (C)
|
Each team will pick 3 or 4 colonies today:
- Set pipette to 3 µl and use a sterile tip to lightly touch a
single colony (do not remove ALL of the colony or gouge the agar!)
Assign an identification symbol to each colony and label the
bottom of the plate under the "spot" -- this plate can be incubated
at 37°C for outgrowth of the individual colonies; you can culture
any "positives" from this plate
- Gently pipet up and down to mix cells with reaction components--the
solution should appear "cloudy"
- After adding all of the reagents, gently tap tubes to
mix
- Take your samples to a thermal cycler (in B05-C.
NOTE: there are only 3 machines; the cycler will be started by the instructor when enough students are ready.
Be certain to record in your notebook the position and labels of your samples and an I.D. of the instrument used.
- Cycling Conditions:
- Cell disruption:
94°C for 10 min
- 30 cycles:
94°C for 30 sec (denaturation)
50°C for 30 sec (annealing)
68°C for 90 sec (extension)
- Final extension:
68
°C for 5 minutes to complete the run
- HOLD at 4°C indefinitely
Agarose gel analysis of PCR products
- Prepare a mini 1% agarose gel with one 8-well comb (use
~40 ml molten agarose) as on Day 2 [pour one gel per team]
- Load 5 µl of each PCR reaction into the gel, alongside 10 µl 1 kb DNA ladder
- Run the gel at 130 V for ~20 min
- Photograph the gel and compare the observed bands to the standards
- Did you get the expected size products?
Expected products for colony screen:
- Negative control = NO bands
- BBa_R0040 =
292 bp
- BBa_E08400 =
1218 bp
- R0040+E0840 (colony PCR) = ~ 1200 bp
Copyright, Acknowledgements,
and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 21 November 2007
Updated 15 October 2014