Arking:JCAOligoTutorial1

This tutorial takes you through the basics of how to design oligos to clone a gene and insert it into a plasmid. At the end of the process, you will have a construction file that describes how all the bits and pieces will be put together, and the sequences of the oligonucleotides you need to order to do your experiment. To do this, you'll need the sequence of the gene you want to clone, the sequence of the plasmid you want to put it in, and a program such as ~ApE to manipulate the sequence.

To get started, let's look at a complete construction file:

 Construction of KanR Basic Part Bca9128
 PCR ca1067F/R on pSB1AK3-b0015           (1055bp, EcoRI/SpeI/DpnI)
 Sub into pSB1A2-I13521                   (EcoRI/SpeI, 2602+946, L)
 Product is pSB1A2-Bca9128  [KanR]
 -------------------------------------
 ca1067F  Forward Biobricking of KanR of pSB1AK3  ccagtGAATTCgtccTCTAGAgagctgatccttcaactc
 ca1067R  Reverse Biobricking of KanR of pSB1AK3  gcagtACTAGTtccgtcaagtcagcgtaatg

This is an example of cloning the gene kanR encoding the kanamycin resistance gene from a plasmid and inserting it into the Biobrick plasmid pSB1A2-I13521.str. The product of the experiment is plasmid JCAseq_pSB1A2-Bca9128. The template for the PCR is JCAseq_SB1AK3-b0015. You can view the sequences for these plasmids by saving the text in these files to your computer.

The format you're seeing is the common GenBank format. Many programs including ApE (A plasmid Editor) can interpret this format and perform the operations described in this tutorial (locating restriction sites and reverse complementing sequences). If you are unable to download a suitable program, these functions can also be performed with the tools at [[1]].

What the construction file is saying to do is set up a PCR reaction with oligonucleotides ca1067F and ca1067R using pSB1A2-I13521 plasmid DNA as template for the reaction. The product of that PCR reaction is 1055 bp, and you should digest it with ~EcoRI, SpeI, and DpnI restriction enzymes. You would in parallel set up a digest of plasmid pSB1A2-I113521 with EcoRI and SpeI which will cut the plasmid into two fragments of sizes 2602 and 946 bp. The "L" means you want to gel purify the large fragment. Upon ligating this fragment to your PCR digest, you will transform and the product of cloning is plasmid pSB1A2-Bca9128.

To understand this further, let's focus on the oligos and template and how they work, so Oligo Design part 1