Cloning Advice in PCR

7/23/2019 Cloning Advice in PCR

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Primer guideline:

Primer guideline:

Short outline: What to prepare ?

Description of restriction enzymes

How to treat internal restriction sites ? Order example: GFPThen:

Get additional advice via E-mail and discuss with organizers

[email protected],

[email protected]

For initial cloning vector ask for plasmid samples:pET His and blue CATG ( a genebank file will then be mailed to you)

Contact: [email protected]

Includes :


2/16

What to prepare I :

What to prepare I :

Order primer ( following slides) : Discuss with organizers

Best including NcoI _ Acc65I sites (see also following slides)

(also possible NcoI - NotI, or BpiI - Acc65I or NotI)

Run PCR

Cut PCR fragment

Clone in basicvector ( Get from course organizers: Prof.Arnt Raae)

Recut and prepare restriction fragment with NcoI/Acc65I

We can start with anything you have in preparation,

but best if you do the following steps before the course starts :

NcoI Acc65Iyour Gene (insert)


3/16

What to prepare II :

What to prepare II :

Our material :

- Tested vectors cut with NcoI/Acc65

and NcoI/NotIYour material :

- Cleaved precloned NcoI/Acc65I or NcoI/NotI

fragment (optimal scenario)

- PCR product, cleaved- Primers and source DNA


4/16

GCTTCGCTTC CCATGATG GGGCGC

CGAAg

CGAAg g t a c

g t a c ccgc

cg

cutcut

NcoI primer

NcoI primerCCATGG = recognition site, which includes ATG start codon Create longer oligo at 5- end, because enzyme cuts more efficient

Start with a G in the triplett coding for 2nd aa to be able to create NcoI site

Options:

Gly GGX

Ala GCX

or Asp or Glu codons

ATGis coding Met

CCATGATG GGGCGC

c cgc cg

overhangoverhang


5/16

55!!GCTTGGCTTG GTAC CGTAC CTTATTAxxxx

33!!CGAA c c aCGAA c c a tgt

g g

g aataat yy

yy

cutcut

Acc65I primer

Acc65I primerGGTACC Create longer oligo at 5 because enzyme cuts more efficient

You have to introduce a stop codon [optimal: TAA] after last aminoacid coding triplet

Remember: Reverse primer is in rev-complementary orientation TTA is the representation of the stop codon in this orientation

GTACGTAC C TTAxxC TTAxx

gg aataatyyyy

overhangoverhang

Stop codon


6/16

55!!GCTTGCGCTTGC GGCC GCGGCC GCTTATTAxxxx

33!!CGAAgc c cCGAAgc c c gggg cgcg aataat yyyy

cutcut

NotI primer

NotI primerGCGGCCGC create longer oligo at 5 because enzyme cuts more efficient

You have to introduce stop codon [optimal: TAA] after last aminoacid

Remember: Reverse primer is in rev-complementary orientation TTA is the representation of the stop codon in reverse orientation

GGCC GCGGCC GCTTAxxTTAxx

cgcg aataatyyyy

overhangoverhang

Stop codon


7/16

If you have internal sites of proposed

enzymes

Check out use of BpiI or similar enzymes: see following

slides

Use of outside non-cutters and initial cloning , then partial

digest with NcoI/Acc65I

NcoI Acc65IBpiI BpiI

NcoI Acc65IEcoRI

HindII

InternalNcoI

Internal

NcoI


8/16

GAAGACGAAGAC TCTC CC ATGATGGG

CTTCTG AG c t a cCTTCTG AG c t a c cccc

g gg g tactac

CATGGCATGG

c

c

BpiIBpiI

cutcut

sequencesequence

recognitionrecognition

Use ofUse of BpiIBpiICreation of NcoI or any overhang :

Enzyme has 2 domains (DNA binding + nuclease), which either :

Recognizes a specific DNA sequence ( Bpi:Recognizes GAAGAC)

Cuts at a specific distance any sequence with creation of a defined overhang:

BpiI : Creates 4 bp overhang 2 bp distant from last recognition base

Separation of recognition and cutting site

Internal NcoI site wont be recognized and cut by BpiI

44 bpbp

overhangoverhang


9/16

Use ofUse of BpiIBpiI: Primer: Primer

Bpi I primer design to create :

NcoI overhang :

Met Gly

CGTTC GAAGACTC CATG GGC XXXXXXXXX

Acc65I overhang :

Stop

CGTTC GAAGACTG GTAC C TTAYYYYYYYY


10/16

Primer design: Create annealing of primers to sourcePrimer design: Create annealing of primers to source

DNA withDNA with ca. 60ca. 6000C full matching annealing temperatureC full matching annealing temperature

>GFP sequence726 bases.

AAAGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGAC

GGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTAC

GGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACC

CTCGTGACCACCTTCGGCTACGGCCTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAG

CAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTC

TTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTG

GTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCAC

AAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAAC

GGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCC

GACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCAC

TACCTGAGCTACCAGTCCGGCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTC

CTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAG

G/C each 4 deg

A/T each 2 deg


11/16

Primer design: AddPrimer design: Add

start ATG / TAA stopstart ATG / TAA stop codoncodon

>GFP sequence + start and stop codonATGAAAGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTG

GACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACC

TACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCC

ACCCTCGTGACCACCTTCGGCTACGGCCTGCAGTGCTTCGCCCGCTACCCCGACCACATG

AAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATC

TTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACC

CTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGG

CACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAG

AACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTC

GCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAAC

CACTACCTGAGCTACCAGTCCGGCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATG

GTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAG

TAA

Start ATG (Met)

Stop TAA


12/16

Primer design: Add restriction sitesPrimer design: Add restriction sites

>GFP sequence (start+stop) + add. Glycinecodon to create NcoI site

CCATGGGCAAAGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCG

AGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATG

CCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCT

GGCCCACCCTCGTGACCACCTTCGGCTACGGCCTGCAGTGCTTCGCCCGCTACCCCGACC

ACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCA

CCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCG

ACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCC

TGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGC

AGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGC

AGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTACCAGTCCGGCCTGAGCAAAGACCCCAACGAGAAGCGCGATC

ACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGT

ACAAGTAAGGTACC

NcoI CCATGG, you need G starting triplet;

add 1 glycine codon GGC

Acc65I GGTACC


13/16

Primer design: Add bases forPrimer design: Add bases for

optimal restriction enzyme cutoptimal restriction enzyme cut

>GFP sequence (start+stop) + add. Glycine

codon to create NcoI site

GCTTCCATGGGCAAAGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTG

GTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGC

GATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTG

CCCTGGCCCACCCTCGTGACCACCTTCGGCTACGGCCTGCAGTGCTTCGCCCGCTACCCC

GACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAG

CGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAG

GGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAAC

ATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGAC

AAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTG

CCCGACAACCACTACCTGAGCTACCAGTCCGGCCTGAGCAAAGACCCCAACGAGAAGCGC

GATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAG

CTGTACAAGTAAGGTACCAAGC

NcoI GCTTCCATGG

Acc65I GCTTGGTACC


14/16

Primer design: ExtractPrimer design: Extract oligosoligos

>GFP sequence (start+stop) + add. Glycine

codon to create NcoI site

GCTTCCATGGGCAAAGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTG

GTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGC

GATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTG

CCCTGGCCCACCCTCGTGACCACCTTCGGCTACGGCCTGCAGTGCTTCGCCCGCTACCCC

GACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAG

CGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAG

GGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAAC

ATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGAC

AAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGC

GTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTG

CCCGACAACCACTACCTGAGCTACCAGTCCGGCCTGAGCAAAGACCCCAACGAGAAGCGC

GATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAG

CTGTACAAGTAAGGTACCAAGC

5 oligo NcoI GCTTCCATGGGCAAAGTGAGCAAGGGCGAGG

3 oligo Acc65I ATGGACGAGCTGTACAAGTAAGGTACCAAGC


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Primer design: OrderPrimer design: Order oligosoligos and run PCRand run PCR

GFPsense GCTCCATGGGCAAAGTGAGCAAGGGCGAGG

GFPrev GCTGGTACCTTACTTGTACAGCTCGTCCAT

Tested PCR conditions with Taq polymerase and 50 ng source plasmid:

95 deg 2 min denaturation

7 cycles

95 deg 20 sec

54 deg 20 sec

72 deg 30 sec

20 cycles95 deg 20 sec

58 deg 20 sec

72 deg 30 sec

72 deg 10 min final extension

GFP sense

GFP rev

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Cloning Advice in PCR