1. Primer Design and Sequencing
ChE

2. General Rules Sterile technique!!
Report any problems to TAs ASAP (and in advance if possible)
Supply concerns (plates, media, tips, tubes, etc.)
Bad reagents
Take good notes
This is critical for troubleshooting; help us help you.
Cleanliness
Keep enzymes (2X Pfu, 2X Paq, Restriction Enzymes, Ligase, Phosphatase) on ice and put them away immediately after using them.
Double-check your work area before leaving
Tips, etc in Biotrash
Dump liquid waste in fume hood
Gels in trash, pour running buffer into the bottle, rinse / dry running apparatus
Ask if everyone wants their own media bottles?

3. Primer Design

4. Denaturation Annealing
Primers
are short synthetic ssDNA or oligonucleotides that serve as starting points for DNA synthesis
“Forward” Primer:
5’ – GCCAGGAGTGAAACGATG – 3’
pYPET Template DNA:
5’ – GCCAGGAGTGAAACGATGTCTAAAGGTGAAGAATTATTCACTGGTGTTGT — 3’
3’ – CGGTCCTCACTTTGCTACAGATTTCCACTTCTTAATAAGTGACCACAACA — 5’
Denaturation Annealing
5’ – GCCAGGAGTGAAACGATG — 3’
3’ – CGGTCCTCACTTTGCTACAGATTTCCACTTCTTAATAAGTGACCACAACA — 5’
Elongation
5’ – GCCAGGAGTGAAACGATGTCTAAAGGTGAAGAA — 3’
3’ – CGGTCCTCACTTTGCTACAGATTTCCACTTCTTAATAAGTGACCACAACA — 5’

5. Denaturation Annealing
Primers
are short synthetic ssDNA or oligonucleotides that serve as starting points for DNA synthesis
“Reverse” Primer:
5’ – GCCACCTTGGCCTTAGTG – 3’
3’ – GTGATTCCGGTTCCACCG – 5’
pYPET Template DNA:
5’ – TGAATGAATTGTACAAACACCACCACCACCACCACTAAGGCCAAGGTGGC — 3’
3’ – ACTTACTTAACATGTTTGTGGTGGTGGTGGTGGTGATTCCGGTTCCACCG — 5’
Denaturation Annealing
5’ – TGAATGAATTGTACAAACACCACCACCACCACCACTAAGGCCAAGGTGGC — 3’
3’ – GTGATTCCGGTTCCACCG — 5’
Elongation
5’ – TGAATGAATTGTACAAACACCACCACCACCACCACTAAGGCCAAGGTGGC — 3’
3’ – GTGGTGGTGGTGGTGGTGATTCCGGTTCCACCG — 5’

6. Guidelines for Primer Design
Length
Melting Temperature
%GC Content
Primer pair (length and Tm)
3’ GC Clamp
Extra nucleotides for restriction enzymes
No secondary structures
Avoid cross-homology

7. Guidelines: Length Binding region (18-25 base pairs)
A tradeoff between specificity and annealing
Oligonucleotide synthesis are 99% efficient
Max primer length will be 60bp
Length
% of Primers w/Correct Sequence
10 bases
20 bases
30 bases
40 bases
50 bases
(0.99)10 = 90.4%
(0.99)20 = 81.8%
(0.99)30 = 74.0%
(0.99)40 = 66.9%
(0.99)50 = 60.5%

8. Guidelines: Melting Temperature (Tm)
Tm is the temperature at which 50% of the primer-target duplex dissociatess
Related to annealing temperature (Ta) for PCRs
Aim for Ta = 55-60°C, which corresponds to Tm = 60-65°C
Many online calculators
Built-in to plasmid editors
Online:
GC content 40%-60%
Can share PCR blocks if using the same PCR program!

9. Guidelines: Primer Pair
Forward and reverse primers should have similar length and Tm to:
Avoid preferential amplification
Simplify PCR optimization
Aim for:
Differences in length < 3 bases
Differences in temperature < 3°C
Example, pYPet primers:
5’ – GCCAGGAGTGAAACGATG – 3’
Forward Primer: 18nt, Tm = 53.5°C
5’ – GCCACCTTGGCCTTAGTG – 3’
Reverse Primer: 18nt, Tm = 56.2°C
|Δlength| = 0
|Δtemperature| = 2.7°C

10. Guidelines: Other Considerations
3’ GC Clamp
The presence of a G or C at the 3’ end promotes correct binding due to the stronger hydrogen bonding of G and C bases
Avoid repeats
Misprime
Errors in synthesis
Extra nucleotides for restriction enzyme (endonucleases) digestions
Restriction Endonuclease
DNA
PDB: 1RVA

11. pZE_Plac-YPet serves as both your template DNA and plasmid backbone
General Workflow
Design primers to amplify Promoter and RBS region
Use restriction enzyme (RE) sites (BamH1 and EcoR1 or Kpn1) Note: these sites are unique, double digest compatible and give ‘sticky ends’
Create designed plasmid through ligation at RE sites
BamHI
NheI
EcoRI
KpnI
HindIII
CRP
Plac
LacO
RBS
YPet

12. Possible Cloning Strategies
RBS Modifications
EcoRI… NNN… 1
CRP
Plac
LacO
RBS
YPet
Promoter Modifications 2
NheI… NNN…
CRP
Plac
LacO
RBS
YPet
BamHI… NNN… 3
CRP
Plac
LacO
RBS
YPet

13. Sequencing

14. How does sequencing work?
Sequencing primer is located ~100bp upstream of segment to be sequenced
Sequencing results are not accurate within the first 100bp region
Accuracy also tapers off after bp
Plasmid DNA sample

15. Sequencing Plasmid DNA
Miniprep overnight culture of cells harboring desired plasmid
Measure concentration of plasmid DNA
Check A260/280
Verify samples are plasmid DNA
Carefully label tubes for samples to be sent for sequencing
400 ng of plasmid
10 µL of 10 μM sequencing primer
Prepare online form
Enclose tubes and printout in a plastic bag
Dropbox will be located outside of Braun 16/17
Pickup is at 1pm

16. But before you sequence…
Verify plasmid DNA is plasmid DNA and contains insert of correct length
Colony PCR
Restriction mapping
DNA gel electrophoresis
+Insert
No Insert
DNA gel

17. Go to http://sequencing.retrogen.com

18. Home Screen

19. Submit sequencing requests

20. Home Screen

21. Results, next morning by 10am

22. DNA editing software Key features:
restriction site recognition, DNA annotations
ApE (A plasmid Editor)
OS X and Windows
pDRAW32
Windows only
Go through ApE with them