Team:Heidelberg/Notebook/miRNA Kit/August
From 2010.igem.org
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* Digestion of the pSB1C3 vector amplified via Phusion HF PCR Mastermix (annealing temperature 61 °C) with EcoRI/PstI for 1 h @ 37 °C. After heat inactivation, 1 ul of DpnI was added to the restriction mix an digestion performed for another hour. The product was SAP (dephosphorylation) or not SAP treated this time. Furthermore, the hRluc, Luc2, CMV_TetO2, sv40 and FRT site PCR products (see 08/27/2010) were digested EcoRI/PstI as well. | * Digestion of the pSB1C3 vector amplified via Phusion HF PCR Mastermix (annealing temperature 61 °C) with EcoRI/PstI for 1 h @ 37 °C. After heat inactivation, 1 ul of DpnI was added to the restriction mix an digestion performed for another hour. The product was SAP (dephosphorylation) or not SAP treated this time. Furthermore, the hRluc, Luc2, CMV_TetO2, sv40 and FRT site PCR products (see 08/27/2010) were digested EcoRI/PstI as well. | ||
* Ligation: each PCR fragment was ligated into digested vector backbone pSB1C3. 50 ng of the non-treated and 100 ng of SAP treated vector were used in the ligation reactions. Amount of insert was calculated to be 2 fold (large fragment Luc2 and hRluc) or 3-5 fold of the molar vector amount. Ligation was performed for 1.5 h @ room temperature. The ligation was analyzed on a 1 % agarose gel, run for 35 min @ 100 V. The non-treated vector is indicated via number 1 (i.e. Luc2.1) the SAP-treated vector with nr. 2 (i.e. Luc2.2). As control, the digested insert fragment PCR products and digested vectors were loaded on the gel as well. For hRluc, Luc2 and CMV_TetO2 correct product bands are clearly visible. For sv40, that is not the case. The FRT site is to short, to be able to distinguish clearly between vector fragment and ligated Vector with Insert. Subsequently, a transformation of all 10 ligation reactions into Top10 cells was performed according to the standard transformation protocol. | * Ligation: each PCR fragment was ligated into digested vector backbone pSB1C3. 50 ng of the non-treated and 100 ng of SAP treated vector were used in the ligation reactions. Amount of insert was calculated to be 2 fold (large fragment Luc2 and hRluc) or 3-5 fold of the molar vector amount. Ligation was performed for 1.5 h @ room temperature. The ligation was analyzed on a 1 % agarose gel, run for 35 min @ 100 V. The non-treated vector is indicated via number 1 (i.e. Luc2.1) the SAP-treated vector with nr. 2 (i.e. Luc2.2). As control, the digested insert fragment PCR products and digested vectors were loaded on the gel as well. For hRluc, Luc2 and CMV_TetO2 correct product bands are clearly visible. For sv40, that is not the case. The FRT site is to short, to be able to distinguish clearly between vector fragment and ligated Vector with Insert. Subsequently, a transformation of all 10 ligation reactions into Top10 cells was performed according to the standard transformation protocol. | ||
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{{:Team:Heidelberg/Single_Bottom}} | {{:Team:Heidelberg/Single_Bottom}} |
Revision as of 00:06, 27 October 2010
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