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The position of the restriction enzymes (NdeI and XhoI) relative to the T7/lac promoter is clearly indicated… but so are the base pair cut sites! Woohoo!!!!!! Note the numbers in brackets beside each restriction enzyme.

Instead, we can take a shortcut based on the information provided in the original, pET26b circular plasmid map shown above. This process for determining DNA fragment sizes works well, but it is time-consuming. We can physically count the base pairs in the yellow highlighted area (there are 128 bps) before we reach the first “c” in the XhoI DNA recognition site, which cuts at exactly bp 5198. If we count base pairs (using the bp numbers on the right as a guide) we can see that the NdeI cuts at exactly the 5070 bp position (remember, we report the bp position relative to the sense strand only). This is to standardize our reporting system, however, we need to keep in mind that we are cleaving both strands of the DNA using restriction enzymes NdeI and XhoI. We typically report the base pair sizes relative to the sense strand (5’ to 3’) only. We can count the number of base pairs (bp) in this area to determine the fragment size cleaved out of the plasmid. The yellow highlighted area contains the DNA fragment cleaved out following NdeI/XhoI digestion of the pET26b plasmid. PET26b plasmid previously digested with NdeI and XhoI =? To answer this question let us zoom in on the pET26b plasmid map and look at the actual sequence surrounding the NdeI and XhoI restriction enzyme DNA recognition sites:

lacI – a gene that expresses the Lac repressor, a protein used to control the T7/ lac promoter.Īnd so, based on this map we can say the following:.

Ori – the origin of replication for the pET26b plasmid.

always on) and is used as a selectable marker for bacterial transformations.

kanR gene, selectable marker – the kanamycin resistance gene is situated downstream of a constitutive promoter (i.e.

Why? Because this His 6x-tag will be used later for protein purification.

His 6x-tag – this is an engineered tag consisting of the codon for the amino acid histidine repeated six times.

This indicates the bp position of the cut site for each RE. Note the bp position in brackets beside each RE.

NdeI and XhoI – the two Restriction Enzymes (RE) we are using to clone our folA gene downstream of the T7/ lac promoter.

We are cloning our folA gene downstream of this promoter. we control when this promoter is off or on).

the T7/ lac promoter – this is the main event! An inducible promoter (i.e.

The pET26b plasmid is a circular, covalently-closed double-stranded DNA, 5360 bp in length. Note the different features of the plasmid and their base pair (bp) location on the plasmid. This is a map generated from the actual pET26b DNA sequence.

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This is a paid software but it does have a basic, freeware version called SnapGene viewer.

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There are many molecular cloning software programs available, and some are free to use. The sequence can be processed using molecular cloning software. One such location is the AddGene portal: pET26b sequence The pET26b plasmid sequence can be sourced from many locations. OK, let’s unpack this, starting with the pET26b plasmid. coli folA gene (and sequence of the DNA primers used in the PCR).

This question is actually quite complex and requires knowledge of the DNA sequence for both pET26b plasmid and the E. If you wish to learn more about our Team Think Tanks, click here. Please check out Team Think 3: Restriction Enzyme Digests located in the Teacher Resources section.

#ADD PIECES IN TO PLASMID MAPS SNAPGENE VIEWER HOW TO#

Helping students understand how to determine DNA fragment sizes following RE digestion reactions makes for a great Team Think Tank. Chapter 2 background – expected DNA band sizes

Add pieces in to plasmid maps snapgene viewer