Cloning Plan number:______
Plasmid number:
Construct name:
Description:
Project:
Date of cloning:
Lab book ref:
Map of plasmid target:
Oligos
Ordered from:
Oligo 1:
Oligo 2:
Outline of work
Insert preparation:
1: Use oligos shown to amplify gene xyz1 from the E.coli K-12 genome (genomic prep
number 7). A 1.2 kb band is expected.
(insert conditions/data/comments here)
2: Clean up PCR reaction using PCR purification kit, and check concentration and purity
by spec measurement and agarose gel)
(insert conditions/data/comments here)
Vector preparation
1: Digest pUC18 (plasmid prep 42) with BamHI, then dephosphorylate with SAP
followed by heat inactivation at 65°C for 15min.
(insert conditions/data/comments here)
2: Run the pUC18 digest on an agarose gel. A 2.6kb band is expected. In parallel, run a
sample of uncut pUC18 to check that the digest has occurred. Isolate the band using a gel
extraction kit.
(insert conditions/data/comments here)
Ligation and transformation
Insert ligation and transformation conditions and outcome
Clone identification
Number of clones picked, isolation of plasmid DNA and digest or PCR to check for
positive clones.
Quality control checks
Digests
Initial digest
Restriction mapping 1
Restriction mapping 2
Sequencing analysis
Sequenced by:
Primers:
Result:
Protein expression
Host strain:
Induction conditions:
Expected protein band:
Observed protein band:
Activity assay
Enzymes
Expected bands
Obtained bands