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2015

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Geco plasmid construction

1. Polymerase Chain Reaction (PCR)

Use PBX-084 TRE-mCherry-2A-Bla plasmid as template to get blasticidin(Bla) resistance gene.

Primers: Age-Bla-S & Bla-E2A-AS

(without purification), 20μl, 30 cycles

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2. PCR

Use PBX-084 TRE-mCherry-2A-Bla plasmid as template to get 2A sequence.

Primers: E2A-S & E2A-Hind III-AS

(without purification), 20μl, 30 cycles

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3. PCR

Step 1: run 10 cycles without primer to let Bla and 2A link together.

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Step 2：add primers to run 30 cycles.

Primers: AgeI-Bla-S 0.3μM & E2A-HindIII-AS 0.3μM

4. PCR product cycle purification by using E.Z.N.A.® Cycle Pure Kit.

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Geco plasmid construction

1. Digest the PCR purification product with AgeI and HindIII restriction endonuclease(RE).(37 ℃ 2 hours)

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2. Digest the Geco plasmid with BglII restriction endonuclease. (50℃ 3 hours)

632x146px3.Cycle pure the RE digestion product with the kit “xxxxxx”.

3. Cycle pure the RE digestion product with the kit “xxxxxx”.

4. Digest the purification product with HindIII restriction endonuclease. (37 ℃ 3 hours)

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5. Digest the vector plasmid with AgeI restriction endonuclease. (37 ℃ 3 hours)

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6.Add 1.5 μl BclII restriction endonuclease. (50 ℃ 3 hours)

Note: BclI and BglII have the same sticky end.

7. Gel running and Gel extraction to get the insert and vector fragments.E.Z.N.A.® Gel Extraction Kit - Spin Protocol

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Geco plasmid construction

1. Geco,Vector,Bla+2A Ligation with T4 DNA Ligase(Vector:Insert=1:3).

Vector (5.8 kbp): 5.5ng/μl

Bla+2A (0.45kbp): 16.8ng/μl

Geco (1.25kbp): 5.6ng/μl

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Room temperature for 10 minutes.