In this section I am going to demonstrate how to create experiment outline in PowerPoint,
build plasmid maps, and design primers for your cloning project.
I begin each of my cloning projects by making a PowerPoint file with a project outline.
For that I open a new PowerPoint file and save it as pSAT6-Bar, which is the project
name. I change the layout to my favourite, which is "Title Only". And I put the project
name on top.
On this slide I put the name of my starting plasmid, which is pSAT6-MCS. Let's add some
style to it. Then I draw a down arrow, add some style to it as well, and put the name
of the plasmid I am trying to make, which is going to be pSAT6-Bar. For this one I will
use a new style, which is going to be black.
Also on this slide I want to have a name of my PCR template, which is a plasmid called
pFGC 5941. Let's give it some style as well. Also I want to have the names of the primers
I will be using, which is Bar-XhoI-F and Bar-BamHI-R.
I will draw another arrow indicating that PCR product will be inserted into pSAT6-MCS.
And, finally, I will indicate the restriction enzymes I will be using to cut my Vector and
Insert.
A usual cloning project goes for several days and in many cases you will be running several
of those projects at a time. That's why I would recommend printing out this page and
keeping it somewhere nearby, so when you come to your lab in the morning you know exactly
where you left off.
Let's move to the next part of the planning stage, which is building vector maps and designing
primers. The software package I will use in this chapter is Vector NTI from Invitrogen.
In Supplement 3, which is at the end of this video, I will demonstrate how to do exactly
the same vector map design with a free software pDRAW32.
I start with two browser windows, one displaying the sequence of pSAT6-MCS, another - sequence
of pFGC5941. I launch Vector NTI Explorer, which keeps track of all the maps I have in
the database. I create a new database subset, label it as Bar, and click on this icon, which
says New. I name the molecule as pSAT6-MCS, indicate that it is circular, and open Edit
Sequence window. Then I go back to the web browser, select and copy the sequence of pSAT6-MCS,
and paste it in the Edit Sequence window.
Let's look at this sequence in Vector NTI. Vector NTI opens with three panes. On the
top right you see the map, on the bottom is the sequence, and on the top left is the information
about the molecule. If you click on the Restriction/Methylation Folder you will see a list of all restriction
enzymes currently displayed on the map. Now, let's add the features of pSAT6-MCS to this
map.
For that I click on ADD FEATURE, switch to the web browser window and select Feature
View. You can see here a list of all pSAT6 features with their coordinates. Let's transfer
them to our map. In the ADD FEATURE menu I am going to enter feature name, which is 2x35S
Promoter, and starting and ending point. In the Vector NTI I could also pick different
feature types, which reflect how the features are displayed on the map. What I particularly
like about VNTI is that as I am adding those features, I can see them both on the map and
on the sequence.
Now I will proceed adding the rest of the features, namely Multiple Cloning Site, Terminator
and Ampicillin resistance gene. Again, I get all the information about feature locations
from the PubMed Nucleotide file.
pSAT6-MCS map is ready, I am going to save it and move onto building pSAT6-Bar.
I will build pSAT6-Bar by pasting the sequence of the bar gene from PubMed Nucleotide.
First, I locate XhoI and BamHI sites on pSAT6-MCS and remove sequence between them.
Then I will go to the PubMed Nucleotide and locate the sequence of the bar gene. When
I scroll down and look in the annotations I can see that the bar gene is located on
the reverse strand in the positions 373 - 924.
To make life a little simpler, I will use PubMed tool called Change Region Shown. I
enter here the boundaries of the bar gene and click on Update View. Now if I scroll
all the way down you can see that the sequence of the pFGC5941 reduced to a 500 base pair
region, which is the bar gene.
Also, if you have a closer look at it, there are an ATG and a Stop codons on the complimentary
strand of this sequence. So, let's copy the bar gene, go back to Vector NTI and paste this sequence
between XhoI and BamHI sites in pSAT6-MCS.
Because the bar gene is located on the complimentary strand, I need to reverse the pasted sequence,
which can be done by going to File - Molecule Operations - Advanced - Reverse Selection.
Now, once the bar sequence is in place, I can label it as a new Feature, by going
to New Feature, typing the feature name, and selecting the feature type, and clicking OK.
Finally, I save this file as pSAT6-Bar.
Now I am going to design PCR primers for cloning the bar gene into pSAT6-MCS.
First, let's find the start of the bar gene. For the Forward primer, I am going to select
the first 20 nucleotides of bar. You can see the number of selected nucleotides right
here at the bottom of the Vector NTI window.
So, I am going to select the first 20 nucleotides of bar along with XhoI restriction site,
and six nucleotides after it: one, two, three, four, five, six. Those six nucleotides are
necessary for XhoI cleavage close to the end of a PCR fragment. That's it, this is my forward
primer. I am going to copy it and submit it to an oligo synthesis company.
Now I am going to scroll down to the end of bar sequence, locate the last twenty nucleotides, select
those twenty nucleotides along with BamHI site. And add additional six nucleotides for
BamHI cutting close to a fragment end.
Since this is the reverse primer, I need to invert it. For that I go to Analysis - Oligo
Analysis - Thermodynamic Properties and select Complimentary. This is the sequence of my
reverse primer. I am going submit it to an oligo synthesis company along with the sequence
of the forward primer.
If you never built plasmid maps before, you might feel that it is just a hustle that you
would rather avoid. But once you get used to having a clear picture of your construct
and access to every little feature on the map, you would never want to walk in the darkness
again.