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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 and DNA design

Assignments Due

Toward reprogramming bacteria with small molecules and RNA, J. Gallivan (Curr Opin Chem Biol 2007, 11:612-619)
Using movies to analyse gene circuit dynamics in single cells, J. Locke and M. Elowitz (Nat Rev Microbiol 2009, 7(5):383-392)
Primer3 Plus, an enhanced web interface to Primer3, A. Untergasser et al. (Nucleic Acids Research 2007, 35: Web Server issue W71-74)
RibosomalBindingSites.pdf (in OWL-Space Resources)

Discussion

Polymerase chain reaction (PCR)
ApE: A plasmid Editor
Ribosomal binding sites (RBS)

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 do you have that your genetic circuit is working?

Background

Polymerase Chain Reaction

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.

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 7 PCR reactions: 1 negative control; 2 using plasmid DNA (diluted 1:100 in nuclease-free water); 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 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

Annotation of Vector Sequence

Download ApE: A plasmid Editor and use it to find the following features in the sequence for BBa_I13522 (see I13522-GFP.docx uploaded in OWL-Space Resources):

Open reading frames (ORF) for GFP and ampicillin resistance (AmpR) gene: use NEBcutter to locate putative ORF's and use BLAST to identify sequence
Use the Registry of Standard Biological Parts to locate
- Promoter
- Ribosomal binding site (RBS)
- BioBrick Prefix and Suffix

Primer Design

Each team uses Primer3 or Primer3Plus to design primers that will mutate a single nucleotide in the ribosomal binding site (RBS) for production of GFP in BBa_I13522
- Analyze your primers with Sigma-Aldrich OligoEvaluator™
Use NEB Tm Calculator to estimate the optimal annealing temperature for PCR: choose "Phusion Hot Start Flex" and "Phusion Hot Start Flex 2X Master Mix"

Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 21 November 2007
Updated 31 October 2013