Difference between revisions of "Part:BBa K1915003:Experience"
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===Applications of BBa_K1915003=== | ===Applications of BBa_K1915003=== | ||
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| + | The inducible lac promoter + LTNF15 was PCR amplified using master mix, DI water, and LTNF primers we got synthesized. We denatured the construct at 95˚C for thirty seconds, annealed at 62˚C for thirty seconds, elongated at 72˚C for thirty seconds and repeated the cycle 25X. Due to the size of the peptide we initially found it difficult to gel extract and purify the inducible lac promoter + LTNF15. To bypass this, we opted to skip the gel extraction step, and directly digest the PCR product with EcoRI and PstI. We had issues with ligating our part into an empty pSB1C3 vector. So we digested RFP in pSB1C3 with EcoRI and PstI and used that vector. Inducible lac promoter + LTNF15 was then ligated with a pSB1C3 vector and then transformed into DH5α competent cells using the heat shock method. One hundred micro liters of the transformation was spread on a LB plate with 35ng/uL chloramphenicol and incubated for 24 hours at 37˚C. The colonies that grew were picked, dipped into a colony PCR reaction containing master mix, DI water and VF VR2 primers and then inoculated in 5mL of LB broth with 5uL of chloramphenicol overnight. A colony PCR gel was run to check which colonies had a transformed recombinant plasmid. A QIAgen miniprep was done to extract the plasmids. Diagnosis gels were run to check if the part was successfully cloned into the vector. | ||
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Colony PCR of transformed inducible lac promoter + LTNF15 in PSB1C3. Lanes 1,2,3,4,5,7,9,10 gave us bands around 500 basepairs which is what was expected for the PCR reaction using our VF2 and VR primers. See the figure 1 above. | Colony PCR of transformed inducible lac promoter + LTNF15 in PSB1C3. Lanes 1,2,3,4,5,7,9,10 gave us bands around 500 basepairs which is what was expected for the PCR reaction using our VF2 and VR primers. See the figure 1 above. | ||
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LTNF has a BamHI restriction site that we utilized to help diagnose if the inducible lac promoter + LTNF15 was in the pSB1C3 vector. The first lane contains the plasmid lacking the BamHI restriction site. Each restriction digest was treated the same. Lanes 2,3,4, and 5 have the inducible lac promoter + LTNF15 cut with BamH1. Bands appear around the expected 2289 bp.See the figure 2. | LTNF has a BamHI restriction site that we utilized to help diagnose if the inducible lac promoter + LTNF15 was in the pSB1C3 vector. The first lane contains the plasmid lacking the BamHI restriction site. Each restriction digest was treated the same. Lanes 2,3,4, and 5 have the inducible lac promoter + LTNF15 cut with BamH1. Bands appear around the expected 2289 bp.See the figure 2. | ||
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[[File:T--Georgia State--inducible lac pro+LT15 restriction digest ECOR1 and PST1.jpeg]] | [[File:T--Georgia State--inducible lac pro+LT15 restriction digest ECOR1 and PST1.jpeg]] | ||
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Figure 3. Gel of Inducible Lac Promoter+LTN-15. The above is a gel of the inducible lac promoter+LTN-15 digested with EcoRI and PstI. | Figure 3. Gel of Inducible Lac Promoter+LTN-15. The above is a gel of the inducible lac promoter+LTN-15 digested with EcoRI and PstI. | ||
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Latest revision as of 16:08, 29 October 2016
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Applications of BBa_K1915003
The inducible lac promoter + LTNF15 was PCR amplified using master mix, DI water, and LTNF primers we got synthesized. We denatured the construct at 95˚C for thirty seconds, annealed at 62˚C for thirty seconds, elongated at 72˚C for thirty seconds and repeated the cycle 25X. Due to the size of the peptide we initially found it difficult to gel extract and purify the inducible lac promoter + LTNF15. To bypass this, we opted to skip the gel extraction step, and directly digest the PCR product with EcoRI and PstI. We had issues with ligating our part into an empty pSB1C3 vector. So we digested RFP in pSB1C3 with EcoRI and PstI and used that vector. Inducible lac promoter + LTNF15 was then ligated with a pSB1C3 vector and then transformed into DH5α competent cells using the heat shock method. One hundred micro liters of the transformation was spread on a LB plate with 35ng/uL chloramphenicol and incubated for 24 hours at 37˚C. The colonies that grew were picked, dipped into a colony PCR reaction containing master mix, DI water and VF VR2 primers and then inoculated in 5mL of LB broth with 5uL of chloramphenicol overnight. A colony PCR gel was run to check which colonies had a transformed recombinant plasmid. A QIAgen miniprep was done to extract the plasmids. Diagnosis gels were run to check if the part was successfully cloned into the vector.
Figure 1. Gel of Inducible Lac Promoter+LTNF-15. The above is a gel of the colony PCR of the inducible lac promoter+LTNF-15.
Colony PCR of transformed inducible lac promoter + LTNF15 in PSB1C3. Lanes 1,2,3,4,5,7,9,10 gave us bands around 500 basepairs which is what was expected for the PCR reaction using our VF2 and VR primers. See the figure 1 above.
Figure 2. Gel of Inducible Lac Promoter+LTNF-15. The above is a gel of the inducible lac promoter+LTN-15 digested with BamHI.
LTNF has a BamHI restriction site that we utilized to help diagnose if the inducible lac promoter + LTNF15 was in the pSB1C3 vector. The first lane contains the plasmid lacking the BamHI restriction site. Each restriction digest was treated the same. Lanes 2,3,4, and 5 have the inducible lac promoter + LTNF15 cut with BamH1. Bands appear around the expected 2289 bp.See the figure 2.
Figure 3. Gel of Inducible Lac Promoter+LTN-15. The above is a gel of the inducible lac promoter+LTN-15 digested with EcoRI and PstI.
A restriction digest of the inducible lac promoter + LTNF15 was done using EcoRI and Pst1. The plasmid pSB1C3 served as a control because we knew that the bands produced would be larger in size than the bands produced from our digest of BBa_ K1915003. In lane 1 the plasmid pSB1C3 with RFP was digested with EcoRI and in lane 2 the plasmid pSB1C3 with RFP was digested with EcoRI and PstI. Lanes 4,6,8, and 10 show the inducible lac promoter + LTNF15 around the expected size of 260 bp.See the figure 3 above.
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