Primer design for PCR cloning
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Primer design for PCR cloning. • special case for when the insert is to be cloned. • PCR also used for diagnostic (is gene present) • PCR also used to quantitate gene/transcript copy #. PCR cloning. • TA cloning, utilizing a T or A single base overhang

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Primer design for PCR cloning

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Primer design for pcr cloning

Primer design for PCR cloning

• special case for when the insert is to be cloned

• PCR also used for diagnostic (is gene present)

• PCR also used to quantitate gene/transcript copy #


Primer design for pcr cloning

PCR cloning

• TA cloning, utilizing a T or A single base overhang

formed by Taq polymerase (Thermotoga aquaticus)

• “Tailed Oligo” technique using oligonucleotides having short regions of target gene non-complementarity and encoding restriction sites compatible with cloning sites on the plasmid vector


Primer design for pcr cloning

ABCDE

prepare PCR product

prepare vector

plasmid

PCR product

B

A

restriction

digest

A

B

ligate


Primer design for pcr cloning

Example of a restriction enzyme cutting site

BamHI

5’-XXXGGATCCXXX-3’

3’-XXXCCTAGGXXX-5’

GATCCXXX-3’

GXXX-5’

5’-XXXG

3’-XXXCCTAG

(colored sequence is the overhang)


Primer design for pcr cloning

Oligo design guidelines

•Tm is temp where 50% of oligos are annealed to target

•Tm should be between 55°C and 80°C

•Tm=81.5 + 0.41(%GC) - 675/N-%mismatch (N=length)

•Tm= 4(G+C) + 2(A+T)

•Ta is annealing temp

•annealing temp should be 5°C below Tm

•too high=low product

•too low=non-specific annealing & spurious products


Primer design for pcr cloning

Oligo design guidelines

•primers should have 17 to 28 nt complementarity

•50-80% GC (can be tough)

•not longer than 35-40 base pairs (due to errors in synthesis)

•3’ end should end in at least one G or C (better if there are 2)


Primer design for pcr cloning

Oligo “tails”

• The 5’ tail encodes restriction site, which when cut yields a compatible end to vector

• tail should also have 5-8 nt beyond restriction site for enzyme to “grab onto”

• primer for 5’ end of gene (upstream or forward primer) may encode an ATG

• primer for 3’ end of gene (downstream or reverse primer) may need to encode a stop codon.

•NdeI and NcoI encode ATG in their restriction sites

• pay attention to frame

• don’t use enzyme that is also found in coding sequence of insert!


Primer design for pcr cloning

Restriction enzyme considerations

• many enzymes that have different cleavage sites that yield compatible cohesive ends (tables in NEB catalog)

• e.g. NdeI yields AT overhang compatible with AseI

• for double digests, try to choose enzymes that will function in the same buffer, otherwise do digests sequentially and change/alter buffer for each enzyme


Primer design for pcr cloning

5’ end of lacZ gene

5’ atgaccatga ttacggattc actggccgtc

3’ tactggtact aatgcctaag tgaccggcag

atg acc atg att acg gat tca ctg gcc gtc

M T M I T D S L A V


Primer design for pcr cloning

3’ end of lacZ gene

5’ gctaccatta ccagttggtc tggtgtcaaa aataa 3’

3’ cgatggtaat ggtcaaccag accacagttt ttatt 5’

cag ttg gtc tgg tgt caa aaa taa

Q L V W C Q K *


Primer design for pcr cloning

Software to help you

• Vector NTI (Mac/PC)

• DNA Strider (Mac only)


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