A routine day
A cloning day, step by step
Open the construct, design and check primers, plan the assembly, work out amounts, pick a diagnostic digest and check the clone.
Who it is for A postdoc or technician building a Golden Gate or Gibson construct.
Step by step
1Sequence
Open the construct's file
Opens GenBank, FASTA and plain files, and .dna files read-only. A part it cannot read is refused by name.
2Sequence
Design primers around the target
Primer pairs around your target, within the limits you set.
PCR primer design for a target region, within your template
Limit Checked for one exact product in this template only. A pair is a design, not evidence that it amplifies or is specific across a genome.
3Sequence
Read the kept pairs before you order them
Lists hairpins and dimers below −5 kcal/mol at 37 °C, and the Tm difference.
Primer check: Tm, dimers, hairpins and binding sites
Limit Only those two checks run. Enzyme sites in tails, binding elsewhere in the backbone and rare codons are not checked.
4Sequence
Export the order sheet
Each kept pair as two oligos, with tails, positions and Tm.
Next, in Plasmid
5Plasmid
Plan the assembly
Restriction, Golden Gate or Gibson, with each junction shown. A conflict is refused with the reason.
6Plasmid
Work out the amounts
Picomoles, copies per microlitre and the insert mass for a ligation.
7Plasmid
Pick a diagnostic digest
Ranks digests whose bands, over 5 percent apart, tell correct, empty and reversed constructs apart, with a schematic gel.
8Sequence
Check the clone against the design
Places Sanger reads on the design, with coverage and the state of each junction.
Limit Today a read with 1,000 bases kept after trimming is placed only on circular references of about 22,675 bases or fewer; on a larger construct the check does not place such a read, states why and counts nothing from it, so the check can complete with zero counts. Counts can also differ between runs: a read whose placement cannot be held in the memory available counts nothing.
What this day does not cover
- Maps are capped at 50,000 bases. The gel is a schematic, not a prediction of band spacing.
- The .dna reader is checked on synthetic files only so far.
- Assembly plans are not saved, and primers are not kept as a reusable library.
Who else this suits For molecular biology
For research and education only, and not medical, investment, regulatory or legal advice.