Difference between revisions of "Part:BBa K3764000:Design"
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This part is a modified version of the pUC19 plasmid. We inserted a specialized cassette in its MCS that allows the generation of single T overhangs upon digestion with Eam1105I (AhdI) restriction endonuclease. The reading frame of the LacZalfa fragment is intact so the blue-white screening procedure can be used for the selection of positive clones. Note that the PCR fragments to be cloned in a T-vector must be generated with a thermostable polymerase that shows non-template dependent terminal transferase activity. This results in a preferential addition of a single adenosine (dA) to the 3′-ends of the amplified PCR product. Using proofreading DNA polymerases or enzyme mixes with such enzymes will compromise the TA cloning procedure. In case you use such ingredients, you should purify your PCR product and then treat it with a Taq pol reaction mixture that contains only dA instead of all four dNTPs. | This part is a modified version of the pUC19 plasmid. We inserted a specialized cassette in its MCS that allows the generation of single T overhangs upon digestion with Eam1105I (AhdI) restriction endonuclease. The reading frame of the LacZalfa fragment is intact so the blue-white screening procedure can be used for the selection of positive clones. Note that the PCR fragments to be cloned in a T-vector must be generated with a thermostable polymerase that shows non-template dependent terminal transferase activity. This results in a preferential addition of a single adenosine (dA) to the 3′-ends of the amplified PCR product. Using proofreading DNA polymerases or enzyme mixes with such enzymes will compromise the TA cloning procedure. In case you use such ingredients, you should purify your PCR product and then treat it with a Taq pol reaction mixture that contains only dA instead of all four dNTPs. | ||
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===References=== | ===References=== | ||
Revision as of 14:15, 21 October 2021
Module for generation of a T-vector
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 341
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 335
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
We design the cassette for TA cloning in such a way that the distance between the two Eam1105I (AhdI) recognition sites is minimal. After digestion, the fragment is way too short to be recovered efficiently by the commonly used kits. This eliminates the need for gel purification and simplifies the entire cloning procedure. Moreover, this fragment does not have stop codons in any of its 6 RFs so even if it stays and gets re-ligated in a reversed orientation, the blue-white screening procedure will recognize these clones as empty (blue). Last but not least, if a single T is added to the 5' ends of the used primers that will result in a stop codon formation after ligation. This improves the resolution of the screening procedure since now positive clones are extremely unlicely to develop a blue colouring.
Source
This part is a modified version of the pUC19 plasmid. We inserted a specialized cassette in its MCS that allows the generation of single T overhangs upon digestion with Eam1105I (AhdI) restriction endonuclease. The reading frame of the LacZalfa fragment is intact so the blue-white screening procedure can be used for the selection of positive clones. Note that the PCR fragments to be cloned in a T-vector must be generated with a thermostable polymerase that shows non-template dependent terminal transferase activity. This results in a preferential addition of a single adenosine (dA) to the 3′-ends of the amplified PCR product. Using proofreading DNA polymerases or enzyme mixes with such enzymes will compromise the TA cloning procedure. In case you use such ingredients, you should purify your PCR product and then treat it with a Taq pol reaction mixture that contains only dA instead of all four dNTPs.