1. Department of Chemistry
Cloning 101:
A Primer
Ghosh Lab
University of Arizona
Department of Chemistry

2. Outline Cloning overview pDRAW32 Design Gene Insert Primers
Further considerations (optimization of the process)
Transformation

3. Cloning Overview Insert (your gene) + Functional Plasmid construct
Four main steps in cloning:
Insert synthesis
Restriction enzyme digest
Ligation
Transformation
Insert
(your gene) +
Functional
construct
Plasmid
(vector)

4. Design Overview Functional construct
Steps to follow in designing your cloning experiment:
Design your gene
Design your insert
Pick your enzymes
Check your design
Recheck your design
Functional
construct

5. pDRAW32 Plasmid maps: pDRAW32
All of the important information in one place!

6. pDRAW32 You can look at the sequence in detail Open reading frames
Translation
Restriction sites
Complementary strand

7. Design of the Gene
If you are cloning out of a known plasmid, just use the sequence that you have
Example, the gene we want:
G C D R A S P Y C G
We got this from phage display:
ggctgcgacagggcgagcccgtactgcggt
G C D R A S P Y C G
Phage sequence
Final sequence for the gene of interest:
ggctgcgacagggcgagcccgtactgcggttaa
G C D R A S P Y C G *
Add a stop codon

8. Design of the Gene
If you are designing the gene from scratch, keep in mind codon usage
Not all codons are created equal
Un-optimized codons could lead to lower expression levels
The codon usage reflects levels of tRNA available in E. Coli
Pay attention to the stop codons too (XL1-Blues read through TAG {amber stop codon} 20% of the time)

9. What if we don’t have the DNA sequence?
Design from scratch! (don’t forget about codon usage)
or preferably…

10. Choice of Restriction Sites/Enzymes
Once you have your gene, you need to design a way to get it into your plasmid
Endonucleases (or restriction enzymes) are enzymes which cut DNA at specific internal recognition sequences
Compare to exonucleases, which cut from one end
You must choose restriction sites that are available in the plasmid you are cloning into
They must not appear in your gene (silent mutation can remove unwanted sites in your designed gene)

11. Really Important Factors to Remember When Choosing Restriction Enzymes
Restriction sites must exist only once in your plasmid
They must be in the correct position relative to the purification tag
Restrictions sites usually add extra residues to your gene product; make sure they are compatible with your peptide/protein
Some restriction sites are sub-optimal for cloning
Blunt end sites
dam and dcm methylation-affected enzymes

12. Blunt vs Sticky Ends Most common restriction enzymes “sticky ends” + +
GATCCGGGCTGCAAGCGGTTAAG
Digestion
AATTCTTAACCGCTTCCAGCCCG
AGCCAG GATCCGGGCTGCAAGCGGTTAAG AATTCGTCGAC
GTCGACG AATTCTTAACCGCTTCCAGCCCG GATCCTGGCT +
GATCCTGGCT
AGCCAG
AATTCGTCGAC
GTCGACG
“Sticky ends”: 5’ or 3’ over-hangs that allow the DNA to anneal even though it is not covalently bound
Help with the next step: ligation
Digestion
ATCGGGCTGCAAGCGGTTAACAG
AGCCAGAT ATCGGGCTGCAAGCGGTTAACAG CTGGTCGAC
CTGTTAACCGCTTCCAGCCCGAT
GTCGACCAG CTGTTAACCGCTTCCAGCCCGAT ATCTGGCT +
AGCCAGAT
CTGGTCGAC
Blunt-end restriction enzymes
ATCTGGCT
GTCGACCAG
No sticky ends

13. dam Methylation
Dam methylase
Dam methylase puts a methyl group on the nitrogen of 6th position of adenosine at the site: GATC
All of the E. Coli that we use generate DNA with dam methylation
Some enzymes only cut dam methylated DNA: eg DpnI
Some enzymes do not cut dam methylated DNA: eg XbaI

14. dcm Methylation
Dcm methylase
Dcm methylase puts a methyl group on the carbon of 5th position of cytidine at the site: CCAGG and CCTGG
The enzyme we use most that can be affected by dcm methylation is SfiI
XL1-Blues and BL21s are both Dcm+

15. Design of the Insert
Once you have your restriction enzymes chosen, it is time to design the final complete gene
The multiple cloning site (or whatever plasmid you are cloning into) should already have the 5’ portion of the gene intact (i.e. RBS, spacer, Met)
Sequences must be in frame
NcoI
BtgI
51 CTTTAATAAG GAGATATACC ATGGGCAGCA GCCATCACCA TCATCACCAC
M G S S H H H H H H
SacI AscI SbfI SalI NotI
BamHI EcoRI EcoICRI BssHII PstI AccI HindIII
101AGCCAGGATC CGAATTCGAG CTCGGCGCGC CTGCAGGTCG ACAAGCTTGC
S Q D P N S S S A R L Q V D K L A

16. Design of the Insert Multiple cloning site
71 ATGGGCAGCAGCCATCACCATCATCACCAC
M G S S H H H H H H
SacI AscI SbfI SalI
BamHI EcoRI EcoICRI PstI AccI HindIII
101AGCCAGGATCCGAATTCGAGCTCGGCGCGCCTGCAGGTCGACAAGCTTGC
S Q D P N S S S A R L Q V D K L A
The gene we want:
ggctgcgacagggcgagcccgtactgcggttaa
G C D R A S P Y C G *
Be aware of the amber stop codon: TAG
BamHI PstI
AGCCAGGATCCGAATTCGAGCTCGGCGCGCCTGCAGGTCGACAAGCTTGC
S Q D P N S S S A R L Q V D K L A
G C D R A S P Y C G *
ggctgcgacagggcgagcccgtactgcggttaa
AGCCAGGATCCGggctgcgacagggcgagcccgtactgcggttaaCTGCAGGTCGACAA

17. Design of the Insert Always check and re-check your sequence!
ATGGGCAGCA GCCATCACCA TCATCACCAC
AGCCAGGATCCGggctgcgacagggcgagcccgtactgcggttaaCTGCAGGTCGACAA
Translate the whole gene
atgggcagcagccatcaccatcatcaccacagccaggatccgggctgcgacagggcgagc
M G S S H H H H H H S Q D P G C D R A S ccgtactgcggttaactgcaggtcgacaa
P Y C G - L Q V D
Everything looks good: in frame the whole way!

18. Design of the Insert The wrong way to do it:
AGCCAGGATCC ggctgcgacagggcgagcccgtactgcggttaaCTGCAGGTCGACAAGCTT
The gene is just inserted after the restriction site, which is out of frame with the plasmid-encoded start-codon/His-tag
atgggcagcagccatcaccatcatcaccacagccaggatccggctgcgacagggcgagcc
M G S S H H H H H H S Q D P A A T G R A cgtactgcggttaactgcaggtcgacaagctt
R T A V N C R S T S
Frame shifted = garbage!
**Some plasmids, for whatever reason, have restriction sites out of frame with the translated gene**

19. Finishing Touches
Restriction enzymes need 5’ and 3’ base pairs to cut properly
NEB has a reference guide for specific enzymes (see link below)
A good rule of thumb is 6 base pairs after the recognition site
Inserting a GC “clamp” at the end and beginning of the sequence is also a good idea
atgggcagcagccatcaccatcatcaccacagccaggatccgggctgcgacagggcgagc
M G S S H H H H H H S Q D P G C D R A S ccgtactgcggttaactgcaggtcgacaa
P Y C G - L Q V D
Final gene, polished and ready to go:
gccagccaggatccgggctgcgacagggcgagcccgtactgcggttaactgcaggtcgacgc
S Q D P G C D R A S P Y C G - L Q V D

20. Design of the Primers + Insert Vector
Once the insert is designed correctly, the next step is designing primers to order from IDT, based on insert synthesis strategy
Three main strategies towards insert synthesis:
PCR amplification
Klenow extension of overlapping primers
Complimentary full-length primers +
Insert
Vector

21. PCR Amplification of Insert from an Existing Gene
The most common method of insert synthesis
Necessitates a pre-existing construct
Extra restriction sites and/or amino acid residues can be added on each side of the gene
Internal mutations are more difficult
Insert

22. PCR Synthesis of Insert
PCR amplification from overlapping primers
No pre-existing construct is needed
PCR products messy, possibly making subsequent rxns difficult
Good for inserts >150 bp
F1: 10x 5’ 3’
F2: 1x 5’ 3’ 3’ 5’
R1: 1x 3’ 5’
R2: 10x
Insert
Full-length insert should still be the major product

23. Klenow Extension of Overlapping Primers
Two primers that are complimentary in their 3’ region are designed (overlap  15bp)
Extended to full length by the Klenow fragment of DNA Polymerase I
Useful if insert is 50 to 150 bp 5’ 3’ 3’ 5’
Insert 5’ 3’
Klenow
Klenow fragment: retains 3’ to 5’ polymerase activity, but does not have exonuclease activity

24. Complimentary Full-Length Primers
The simplest approach
Order two primers that compliment each other
Mix the two primers, heat, and aneal slowly (to ensure proper base-pairing)
Feasible if the total insert size is < 60 bp 5’ 3’
Anneal
Insert 3’ 5’

25. Designing Primers to Order
Once the insert synthesis technique is decided, primer design is fairly straight-forward
Forward primers:
Assess necessary overlap and copy the sequence from your designed gene, along with extra 5’ sequence
Reverse primers:
First, design exactly as if it were a forward primer: Copy necessary overlap and extra 3’ sequence from your designed gene
Once all this is in place, use pDRAW32 sequence manipulator to calculate the reverse compliment
Order the pDRAW32 calculated sequence directly

26. Cloning Out an Existing Gene
In the example mentioned previously, we would normally use full length overlapping primers, but let’s look at the more common case of having a preexisting gene:
Preexisting gene:
Overlap
tgcggcccagccggccatgggctgcgacagggcgagcccgtactgcggtggaggcggtgctgcagcgc
A A Q P A M G C D R A S P Y C G G G G A A A +
Goal gene:
gccagccaggatccgggctgcgacagggcgagcccgtactgcggttaactgcaggtcgacgc
S Q D P G C D R A S P Y C G - L Q V D
Extra sequence from gene design
gccagccaggatccgggctgcgacagg
ccgtactgcggttaactgcaggtcgacgc
Forward Primer:
Design of Reverse Primer:

27. Ordering Primers & Forward primer to order: Design of Reverse Primer:
gccagccaggatccgggctgcgacagggcgagcccgtactgcggttaactgcaggtcgacgc
S Q D P G C D R A S P Y C G - L Q V D
Forward primer to order:
gccagccaggatccgggctgcgacagg
Design of Reverse Primer:
ccgtactgcggttaactgcaggtcgacgc
&
Reverse primer to order:
GCGTCGACCTGCAGTTAACCGCAGTACGG
Now we can order the primers:

28. Vectors and Bacteria Strains
An important thing to think about before you start cloning: What vectors/E Coli should I use?
Vector
Promoter
E Coli strains we use
pQE-30
T5 promoter
XL1-Blue: mostly good for DNA isolation/phage display
M15(pREP4): tighter regulation of the lac suppressor
pMAL
Ptac promoter
pCANTAB-5E
Plac promoter
pET-Duet
pRSF-Duet
T7 lac promoter
(An E. Coli strain with phage T7 RNA polymerase is necessary)
BL-21: Protease deficient, stable to toxic proteins, and contains the T7 RNA polymerase gene

29. lac Expression Regulation
RNA polymerase
lac site
Promoter
RBS
ATG- your gene X
IPTG (or lactose, etc)
lac repressor
lac site
Promoter
RBS
ATG- your gene
IPTG
Transcription
lac site
Promoter
RBS
ATG- your gene
mRNA

30. Purification Tags and Selection (Anti-biotic Resistance)
Anti-biotic resistance (working concentration)
Ampicillin (100g/mL)
Kanamycin (35g/mL)
Tetracycline HCl (10g/mL)
Chloramphenicol (170g/mL in ethanol)
Purification Tag
His-tag (nickel agarose resin)
Maltose Binding Protein (amylose resin)
Glutathione S-Transferase (glutathione resin)

31. Digestion of Insert and Vector
Digest with the same restriction endonucleases
Optional (recommended) step:
Treat the plasmid DNA with Antarctic phosphatase
Decreases the background by stopping self-ligation of singly cut plasmid and background re-ligation

32. Ligation of the Insert into the Vector+
Ligation covalently attaches the vector and the insert via a phosphodiester bond (5’phosphate and 3’ hydroxyl of the next base)

33. Antarctic Phosphatase and Ligation
Antarctic Phosphatase cleaves this phosphate, disallowing self-ligation
The insert still has the 5’ phosphate though

34. Transformation
The functional construct is now ready to be transformed into new E. Coli and grown up
The new DNA isolated from the E. Coli must then be sequenced to make sure that everything worked
Once the sequence is confirmed, we are ready to go!