INSERT PREPARATION VECTOR PREPARATION...DNA End Modifications OR OR OR OR OR INSERT PREPARATION...
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dsDNA intermediate 2 DNA Isolation (Plasmid Purification) DNA Analysis DNA Preparation Vector & Insert Joining DNA Processing DNA End Modifications Traditional Cloning (RE Digestion & Ligation) PCR Cloning (TA & Blunt-End) Restriction Enzyme (RE) Digestion PCR Seamless Cloning (Gene Assembly) LIC (Ligation Independent Cloning) Recombinational (Gateway/Creator/Univector) OR OR dsDNA intermediate + OR OR OR OR Starting Material Starting Material RE Digest Colony PCR Sequencing Transformation Protein Expression Functional Analysis Site-Directed Mutagenesis DNA End Modifications OR OR OR OR OR VECTOR PREPARATION INSERT PREPARATION P P P P OR OR Linear vector, ready for joining P P P P P P P P P P P P P P P P OR OR OR OR OR OR Assembled vector Linear vector Plasmid Multiple cloning site (MCS) A A T T T A A T OR A A OR A A OR A A cDNA Synthesis RNA Recombination sites 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. Dephosphorylation Blunting (Optional) Ligation Phosphorylation (Optional) Cohesive-End Formation by 5´ → 3´ exo Annealing Cohesive-End Formation by 3´ → 5´ exo PCR PCR 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 simultaneously with previous step 30 min. 3 hr., 15 min. – 5 hr., 20 min. 75 min. Gel and Column Purification Ligation Clean Up 15 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 OR Gel & Column Purification 75 min. 1.5 hr. T-addition Ligation RE Digestion Proteinase K Treatment 10 min. 70 min.** Site-Specific Recombination 60 min. Holding vector Endpoint vector PCR products Plasmid Annealed oligos gDNA 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
Transcript of INSERT PREPARATION VECTOR PREPARATION...DNA End Modifications OR OR OR OR OR INSERT PREPARATION...
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
