Hix and RE
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| - | We received oligos of Hix L,C, and R from the Mammoths and annealed them as well as the recombinational enhancer(RE) oligos that also came in last week. We used the annealing buffer found on the Missouri Western Mammoth’s page [http://2006.igem.org/Annealing_Oligos]. The oligos and the annealing buffer were placed in to boiling water. The water was allowed to boil for 5min. After this time the flame was turned off and the oligos and buffer were allowed to cool to bench temp. The inserts were then ligated into plasmids containing<font color='red'> RFP</font color>[http://www.bio.davidson.edu/courses/Molbio/Protocols/ligation.html| (ligation protocol)]. We then transformed cells (JM109) and plated them onto LBamp plates[http://www.bio.davidson.edu/courses/Molbio/Protocols/transformation.html| (transformation protocol)]. After incubating overnight the plates were observed to see if there were any red colonies, which would indicate that the Hix or RE insert was not in the plasmid. The plates were placed into the refrigerator to allow the expression of <font color='red'>RFP</font color> to be become more visible. White colonies were picked from the plate and placed into culture tubes containing 2ml of LBamp. These were then miniprepped and double digested using EcoRI and PstI. Following digestion the plasmids were run in a 2.2% agarose gel. It is very clear that the RE insert is present in the plasmid. | + | We received oligos of Hix L,C, and R from the Mammoths and annealed them as well as the recombinational enhancer(RE) oligos that also came in last week. We used the annealing buffer found on the Missouri Western Mammoth’s page [http://2006.igem.org/Annealing_Oligos]. The oligos and the annealing buffer were placed in to boiling water. The water was allowed to boil for 5min. After this time the flame was turned off and the oligos and buffer were allowed to cool to bench temp. The inserts were then ligated into plasmids containing<font color='red'> RFP</font color>[http://www.bio.davidson.edu/courses/Molbio/Protocols/ligation.html| (ligation protocol)]. We then transformed cells (JM109) and plated them onto LBamp plates[http://www.bio.davidson.edu/courses/Molbio/Protocols/transformation.html| (transformation protocol)]. After incubating overnight the plates were observed to see if there were any red colonies, which would indicate that the Hix or RE insert was not in the plasmid. The plates were placed into the refrigerator to allow the expression of <font color='red'>RFP</font color> to be become more visible. White colonies were picked from the plate and placed into culture tubes containing 2ml of LBamp. These were then miniprepped and double digested using EcoRI and PstI. Following digestion the plasmids were run in a 2.2% agarose gel. It is very clear that the RE insert is present in the plasmid. After running and analyzing the gels it is evident that we have successfuly cloned all of the Hix sites as well as the RE site into vectors. |
Revision as of 13:10, 20 June 2006
We received oligos of Hix L,C, and R from the Mammoths and annealed them as well as the recombinational enhancer(RE) oligos that also came in last week. We used the annealing buffer found on the Missouri Western Mammoth’s page [http://2006.igem.org/Annealing_Oligos]. The oligos and the annealing buffer were placed in to boiling water. The water was allowed to boil for 5min. After this time the flame was turned off and the oligos and buffer were allowed to cool to bench temp. The inserts were then ligated into plasmids containing RFP[http://www.bio.davidson.edu/courses/Molbio/Protocols/ligation.html| (ligation protocol)]. We then transformed cells (JM109) and plated them onto LBamp plates[http://www.bio.davidson.edu/courses/Molbio/Protocols/transformation.html| (transformation protocol)]. After incubating overnight the plates were observed to see if there were any red colonies, which would indicate that the Hix or RE insert was not in the plasmid. The plates were placed into the refrigerator to allow the expression of RFP to be become more visible. White colonies were picked from the plate and placed into culture tubes containing 2ml of LBamp. These were then miniprepped and double digested using EcoRI and PstI. Following digestion the plasmids were run in a 2.2% agarose gel. It is very clear that the RE insert is present in the plasmid. After running and analyzing the gels it is evident that we have successfuly cloned all of the Hix sites as well as the RE site into vectors.
