In silico cloning - Bits.vib.be

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Tutorial 4: Gateway cloning The Workbench allows to perform in silico Gateway cloning, including Multisite Gateway cloning. In the Gateway cloning system your gene of interest is inserted into an entry vector. The entry vector contains attP recombination sites. To insert your gene of interest into the entry vector, you need to create a PCR product with terminal attB sites, using primers containing a 25-base pair attB sequence, plus four terminal G's. This PCR product can then be inserted in the entry vector using the BP reaction, resulting in the formation of an entry clone (purple vector at the top) carrying the gene of interst flanked by attL sites. Once the entry clone has been formed it is available to express your gene in a variety of other destination vectors (red vector on the top) which contain attR sites using an LR reaction. The result is an expression clone (red vector at the bottom) with your gene of interest inserted in the destination vector. The attB’s, attP’s, attL’s and attR’s are the key component of the Gateway system: they are the restriction sites. All entry clones have attL's on both sides of their gene of interest. These attL's are cut to form sticky ends by the Gateway recombination proteins. These sticky ends will then match up with the sticky ends on a destination vector which contains attR restriction sites. This process is called a LR reaction. The BP reaction is done similarly using other restriction sites. Note that the LR reaction creates attB sites again in the expression clone: this makes the Gateway system totally reversible. You can make more entry vectors from the expression clone using a BP reaction, in which the attB sites recombine with attP's to form attL's and attR's. The system has a high transformation efficiency since it uses both antibiotic resistance and the ccdB gene, which yields a product that is toxic to E. coli, to ensure that only correct clones survive. The three tools for Gateway cloning mimic the procedure followed in the lab: 1. attB sites are added to a sequence fragment 2. attB-flanked fragment is recombined in an entry vector (BP reaction) to construct an entry clone 3. target fragment from the entry clone is recombined into a destination vector (LR reaction) to construct an expression clone. For Multisite Gateway cloning, multiple entry clones can be created that can recombine in the LR reaction. To perform these analyses in the CLC Main Workbench, you need to import donor and expression vectors. These can be downloaded from Invitrogen’s web site and directly imported into the Workbench: http://igene.invitrogen.com/products/selector/vectors.
4.1 Add attB sites The first step is to amplify the target sequence with primers including the so-called attB sites. Suppose you want to clone the CDS of ATP8a1. First you open the ATP8a1 mRNA sequence in the Main Area. In the Sequence Settings tab of the Side Panel open the Annotation types settings and deselect Gene. Select the CDS. Right click the CDs and choose Open Annotation in New View. Save the CDS as ATP8a1 CDS and add attB sites to the ATP8a1 CDS sequence fragment in this way: Toolbox | Cloning and Restriction Sites | Gateway Cloning | Add attB Sites
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 and 14: Click Next In this step you specify
Page 15 and 16: Click OK and the sequence will be i
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: Note that the Workbench is validati
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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