In silico cloning - Bits.vib.be

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We come back to our in silico cloning problem. If you have chosen the enzymes that you want to work with (in our case: HindIII and XhoI), close the views of the vector and the mRNA sequence and open the ATP8a1 fwd primer sequence. When it opens, double-click the name of the sequence to make a Selection of the full sequence. If you do not see the whole sequence turn purple, please make sure you have the Selection Tool chosen. If the sequence is selected, right-click and choose Insert Restriction Site Before Selection In the Filter box enter HindIII and click on it. At the bottom of the dialog, add a few extra bases 5' of the cut site (this is done to increase the efficiency of the enzyme).
Click OK and the sequence will be inserted at the 5' end of the primer. Perform the same process for the ATP8a1 rev primer, this time using XhoI instead. This time, you should also add a few bases at the 5' end as was done when inserting the HindIII site to increase to efficiency of the enzyme. Note! The ATP8a1 rev primer is designed to match the negative strand, so the restriction site should be added at the 5' end of this sequence as well (using Insert Restriction Site before Selection). Save the two primers and close the views. 2.3 Simulate PCR to create the fragment Now, we want to extract the PCR product from the template ATP8a1 mRNA sequence using the two primers with restriction sites: Toolbox | Primers and Probes | Find Binding Sites and Create Fragments
Page 1 and 2: Tutorial 1: Primer design In this t
Page 3 and 4: Clicking the dot will select the re
Page 5 and 6: By adjusting the Primer parameters
Page 7 and 8: In the Side Panel of the view conta
Page 9 and 10: Tutorial 2: In silico cloning workf
Page 11 and 12: You can do all kinds of restriction
Page 13: Click Next In this step you specify
Page 17 and 18: fine the minimum number of position
Page 19 and 20: Use the Browse button to select pcD
Page 21 and 22: Next step is to cut the fragment. A
Page 23 and 24: Save the construct shown in the cir
Page 25 and 26: Switch to the fragment sequence. Cl
Page 27 and 28: Note that the Workbench is validati
Page 29 and 30: 4.1 Add attB sites The first step i
Page 31 and 32: This will allow you to choose which
Page 33 and 34: You also get a list of primers. Aga
Page 35 and 36: Next you have to specify the entry
Page 37 and 38: When you have selected your entry c

In silico cloning - Bits.vib.be

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