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Often in a cloning project there is a need to assay the success of each cloning step. Typically, this is done by gel electrophoresis. This tutorial will teach you how to run simulated gels using GCK.
1. Start GCK and open the files construct#6 and construct#7 in your tutorial files folder. Recall that these files represent an hsp70 fragment cloned into a vector in two different orientations. We will differentiate between the two orientations by gel electrophoresis in this tutorial.
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Figure 40: Marking Sites for Orientation Analysis
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2. Bring the construct#6 window to the front by clicking on it or by choosing it from the Window menu. Make sure nothing is selected by clicking once in the DNA -- you should see a blinking insertion point. Choose Construct > Features > Mark Sites. Find BfaI in the list on the left and double-click on it to move it to the list on the right. Now find BsiHKAI and move it to the list on the right. Your dialog should look like Figure 40. Press OK.
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Figure 41: BfaI Sites Marked in Construct#6
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3. You will see a number of sites marked in construct#6. Click someplace in the window background to deselect all sites. Double-click one of the BfaI sites. Double-clicking a site name will select all other sites having the same name. Your window will look like Figure 41. Choose Edit > Copy to copy the selected sites to the clipboard. Notice that you are not copying a DNA sequence, but rather a set of markers.
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Figure 42: Creating a New Gel Window
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4. Choose File > New..., which will bring up Figure 42. Click the Gel button and then type in the name orientation analysis for the new window. Press OK. You will see Figure 43. Along the left side are size markers. The blinking triangle at the top of the window indicates the insertion point -- where the next gel lane will be created.
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Figure 43: An Empty Gel Window
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5. Choose Edit > Paste to paste the site markers into lane 1. You will see Figure 44A. This shows the predicted migration of the restriction fragments on a gel. Click the mouse in this window near the top right corner of the gel, just above the gel tab that is sticking up. This will place the blinking triangle insertion point at that location.
6. Now bring the construct#6 window to the front again and double-click on a BsiHKAI site to select all of these sites. Choose Edit > Copy.
7. Return to the Gel window, make sure the triangle is on the right of the gel and choose Edit > Paste to paste the digest into lane 2. You should see Figure 44B. These are the digests you would get from construct#6, having the hsp70 insert in the clockwise direction. In order to compare these digests with ones from construct#7, which has the hsp70 insert in the counter-clockwise direction, you need to do a similar series of steps on construct#7. So...
8. Repeat the procedure with BfaI and BsiHKAI using construct#7. In the construct#7 window double-click on a BfaI site to select all of those sites. Choose Edit > Copy to copy the selected sites to the clipboard.
9. Go to the orientation analysis gel window and click the mouse at the top of the gel between lanes one and two to place the blinking triangle insertion point at that location. Choose Edit > Paste to paste the site markers into the new lane 2 -- note that the construct#6/BsiHKAI digest is now lane 3.
10. Finally, go back to construct#7, select all the BsiHKAI sites, copy them and paste them into the right edge of the gel to produce Figure 44C.
11. If you would like to add a lane of standards, you can open the file called standards gel in the tutorial files folder to see several standard marker digests. Click on the lane that is of interest to you to select it (it will become highlighted), copy it, and paste it into your orientation analysis gel, just as you did for the digests. If you would like to see what this looks like, open the file called orientation analysis in the tutorial files folder.
12. If you do the actual cloning experiment and run a gel, you should be able to distinguish the orientation of the insert by comparing the real gel to the predicted gel pattern shown in Figure 44C. Open the Illustration called real gel analysis in the Sample Files folder to see a real example of this.
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