dsDNAintermediate 2

DNA Isolation(Plasmid Purification)

DNAAnalysis

DNAPreparation

Vector & InsertJoining

DNAProcessing

DNA End Modifications

Traditional Cloning(RE Digestion & Ligation)

PCR Cloning(TA & Blunt-End)

Restriction Enzyme(RE) Digestion PCRSeamless Cloning

(Gene Assembly)LIC (Ligation

Independent Cloning)Recombinational

(Gateway/Creator/Univector)

OR OR

dsDNAintermediate

+

OR OR OR OR

StartingMaterial

StartingMaterial

RE Digest Colony PCR Sequencing

Transformation ProteinExpression

FunctionalAnalysis

Site-DirectedMutagenesis

DNA EndModifications

OR OROR OR OR

VECTOR PREPARATIONINSERT PREPARATION

P

P

P

P

OR

OR

Linear vector,ready for joining

PP

PPP

P

P

P

P

P

P

P

P

PP

P

OR OR

OR OR

OR

OR

Assembled vector

Linear vector

Plasmid

Multiple cloning site(MCS)

AA

TT

TA

AT

OR

AA

OR

AA

OR

AA

cDNA SynthesisRNA

Recombinationsites

Estimated total time*

60 min. (Standard)5–15 min. (Time-Saver)

10 min.

10 min.

15 min.

60 min. (Standard)5–15 min. (Time-Saver)

90 min. 90 min. 90 min. 90 min. 2 hr.

10 min.

DephosphorylationBlunting(Optional)

Ligation

Phosphorylation(Optional)

Cohesive-EndFormation by 5´ → 3´ exo

Annealing

Cohesive-EndFormation by 3´ → 5´ exo

PCRPCR PCR

Instant – 15 min.

1 hr., 20 min. - 2 hr., 40 min.

Estimated total time 20 min. – 2 hr., 25 min. 3 hr., 45 min.

15 min.

2 hr. – 2 hr., 30 min. 2 hr., 15 min. 2 hr., 45 min.

Occurs simultaneouslywith previous step

30 min.

3 hr., 15 min. – 5 hr., 20 min.

75 min.

Gel and ColumnPurification

Ligation

Clean Up15 min.Clean Up

15 min.

30 min. 30 min.

Clean Up

15 min.Clean Up

15 min.Clean Up

15 min.Clean Up

RE Digest

PCR RE Digestion PCR

Dephosphorylation (Optional)

Clean Up 15 min ORGel & Column Purification 75 min.

1.5 hr.T-addition

Ligation

RE Digestion

Proteinase K Treatment10 min.

70 min.**

Site-SpecificRecombination60 min.

Holding vectorEndpoint vector

PCR productsPlasmid Annealed oligosgDNA cDNA

P

P

60 min. (Standard)5–15 min. (Time-Saver)

* Note that times are based on estimates for moving a gene from one plasmid to another. If the source for gene transfer is gDNA, add 2 hours to calculation for the traditional cloning method. Total time does not include transformation, isolation or analysis.** 70 minutes for recombination occurs on second day