Difference between revisions of "Part:BBa K2607002:Experience"
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how you used this part and how it worked out. | how you used this part and how it worked out. | ||
| − | ===Experiment with BBa_K2607002 and BBa_K592024=== | + | ===Experiment with BBa_K2607002 and BBa_K592024 (iGEM 2018 UI_Indonesia)=== |
We performed experiment to compare BBa_K2607002 with BBa_K592024. <b>Figure 1</b> shows the plan on how we conduct the experiment. | We performed experiment to compare BBa_K2607002 with BBa_K592024. <b>Figure 1</b> shows the plan on how we conduct the experiment. | ||
[[File:T--UI_Indonesia--Figure2Improve.png|thumb|center|<b>Figure 1.</b> Original plan workflow with newly-established (BBa_K2607002) and existing (BBa_K592024) parts of BFP. | [[File:T--UI_Indonesia--Figure2Improve.png|thumb|center|<b>Figure 1.</b> Original plan workflow with newly-established (BBa_K2607002) and existing (BBa_K592024) parts of BFP. | ||
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<b>Gel Electrophoresis Confirmation</b><br> | <b>Gel Electrophoresis Confirmation</b><br> | ||
Upon receiving our “Improved BFP” gBlock from IDT (with length of 1151 basepairs (bp) with SalI and NdeI restriction sites are located at base 244-249 and 266-271, respectively), we performed PCR to amplify it until sufficient quantity (about 100 ng/uL). We then created two restriction digestion reactions, one with NdeI restriction enzyme and one with SalI restriction enzyme. Each reaction comprised of 100 uL PCR-amplified “Improved BFP” gBlock, 6 uL restriction enzyme, 15 uL CutSmart® restriction buffer, and 29 uL nuclease-free water. The reaction was subsequently incubated for four hours at 37<sup>o</sup>C incubator. The digestion products were then run for electrophoresis in 1% agarose gel with tris-acetic acid-EDTA (TAE) buffer 1x, power supply 50 volt for 70 minutes. Visualization was performed with Bio-Rad Gel DocTM XR+ Gel Documentation System. <b>Figure 2</b> shows gel electrophoresis result of PCR-amplified "Improved BFP" gBlock. | Upon receiving our “Improved BFP” gBlock from IDT (with length of 1151 basepairs (bp) with SalI and NdeI restriction sites are located at base 244-249 and 266-271, respectively), we performed PCR to amplify it until sufficient quantity (about 100 ng/uL). We then created two restriction digestion reactions, one with NdeI restriction enzyme and one with SalI restriction enzyme. Each reaction comprised of 100 uL PCR-amplified “Improved BFP” gBlock, 6 uL restriction enzyme, 15 uL CutSmart® restriction buffer, and 29 uL nuclease-free water. The reaction was subsequently incubated for four hours at 37<sup>o</sup>C incubator. The digestion products were then run for electrophoresis in 1% agarose gel with tris-acetic acid-EDTA (TAE) buffer 1x, power supply 50 volt for 70 minutes. Visualization was performed with Bio-Rad Gel DocTM XR+ Gel Documentation System. <b>Figure 2</b> shows gel electrophoresis result of PCR-amplified "Improved BFP" gBlock. | ||
| − | [[File:T--UI_Indonesia--Figure3Improve.png|thumb|center|<b>Figure 2. Gel electrophoresis of restriction of “Improved BFP” gBlock with 1% agarose gel, TAE 1x, 50 V, and 70 minutes. | + | [[File:T--UI_Indonesia--Figure3Improve.png|thumb|center|<b>Figure 2.</b> Gel electrophoresis of restriction of “Improved BFP” gBlock with 1% agarose gel, TAE 1x, 50 V, and 70 minutes. Lane 1 = BioLabs, Inc. 100 bp DNA ladder (#N3231L) with size range 100-1517 bp. Lane 2 = uncut PCR-amplified “Improved BFP” gBlock (expected size 1151 bp). Lane 3 = “Improved BFP” gBlock cut with NdeI. Lane 4 = “Improved BFP” gBlock cut with SalI. |
]] | ]] | ||
At lane 2 consisting of uncut “Improved BFP” gBlock (size 1151 bp), only single band is observed. The band is located between 1000-1200 bp marker at lane 1, which is consistent to the gBlock original size. Upon restriction with NdeI, the expected bands are 271 bp and 880 bp in size, while restriction with SalI will yield expected bands of 249 bp and 902 bp. At lane 3 and 4, there are three bands observed, indicating that the gBlocks were partially digested with respective enzymes, NdeI and SalI. The heaviest bands in lane 3 and 4 were parallel with the band in lane 2, indicating that the bands were uncut gBlocks. The middle bands in lane 3 and 4 were similar in size (around 900 bp in size), representing the larger fragments of digestion products with their respective enzymes. The lightest bands in lane 3 and 4 were also similar in size, located between 200-300 bp marker at lane 1. They represent the smaller fragments of digestion products. The smaller fragment upon NdeI digestion is 271 bp, while upon SalI digestion is 249 bp. The lightest band in lane 3 migrated slightly slower than lightest band in lane 4, denoting larger size of smaller fragment in lane 3 than lane 4, which is consistent to the expected restriction results. | At lane 2 consisting of uncut “Improved BFP” gBlock (size 1151 bp), only single band is observed. The band is located between 1000-1200 bp marker at lane 1, which is consistent to the gBlock original size. Upon restriction with NdeI, the expected bands are 271 bp and 880 bp in size, while restriction with SalI will yield expected bands of 249 bp and 902 bp. At lane 3 and 4, there are three bands observed, indicating that the gBlocks were partially digested with respective enzymes, NdeI and SalI. The heaviest bands in lane 3 and 4 were parallel with the band in lane 2, indicating that the bands were uncut gBlocks. The middle bands in lane 3 and 4 were similar in size (around 900 bp in size), representing the larger fragments of digestion products with their respective enzymes. The lightest bands in lane 3 and 4 were also similar in size, located between 200-300 bp marker at lane 1. They represent the smaller fragments of digestion products. The smaller fragment upon NdeI digestion is 271 bp, while upon SalI digestion is 249 bp. The lightest band in lane 3 migrated slightly slower than lightest band in lane 4, denoting larger size of smaller fragment in lane 3 than lane 4, which is consistent to the expected restriction results. | ||
| + | <br><br> | ||
| + | <b>Ultraviolet (UV) Check</b> | ||
| + | <br> | ||
| + | First, we acquired BBa_K592024 by transforming wild-type <i>Escherichia coli</i> K-12 with plasmid in well 17C of Distribution Kit Plate 1, isolating the plasmid, and performing restriction with EcoRI and PstI restricton enzymes. Our “Improved BFP” and BBa_K592024 were separately cloned into plasmid pQE80L using EcoRI and PstI according to our laboratory protocol. The resultant ligation products were subsequently transformed into wild-type <i>E. coli</i> K-12, along with empty pQE80L as control. Transformation products were spread into Luria-Bertani (LB) agar medium containing ampicillin with ratio 1000:1. On the following day, the fluorescence was assessed qualitatively under ultraviolet (UV) light (<b>Figure 3</b>). | ||
| + | [[File:T--UI_Indonesia--Figure4Improve.png|thumb|center|<b>Figure 3.</b> Colony of transformed wild-type Escherichia coli K-12 with empty pQE80L (yellow arrow), pQE80L with “Improved BFP” (green arrow), and pQE80L with BBa_K592024 (orange arrow) in plasmid pQE80L under ultraviolet (UV) illumination. | ||
| + | ]] | ||
===User Reviews=== | ===User Reviews=== | ||
Revision as of 17:07, 12 October 2018
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how you used this part and how it worked out.
Experiment with BBa_K2607002 and BBa_K592024 (iGEM 2018 UI_Indonesia)
We performed experiment to compare BBa_K2607002 with BBa_K592024. Figure 1 shows the plan on how we conduct the experiment.
Gel Electrophoresis Confirmation
Upon receiving our “Improved BFP” gBlock from IDT (with length of 1151 basepairs (bp) with SalI and NdeI restriction sites are located at base 244-249 and 266-271, respectively), we performed PCR to amplify it until sufficient quantity (about 100 ng/uL). We then created two restriction digestion reactions, one with NdeI restriction enzyme and one with SalI restriction enzyme. Each reaction comprised of 100 uL PCR-amplified “Improved BFP” gBlock, 6 uL restriction enzyme, 15 uL CutSmart® restriction buffer, and 29 uL nuclease-free water. The reaction was subsequently incubated for four hours at 37oC incubator. The digestion products were then run for electrophoresis in 1% agarose gel with tris-acetic acid-EDTA (TAE) buffer 1x, power supply 50 volt for 70 minutes. Visualization was performed with Bio-Rad Gel DocTM XR+ Gel Documentation System. Figure 2 shows gel electrophoresis result of PCR-amplified "Improved BFP" gBlock.
At lane 2 consisting of uncut “Improved BFP” gBlock (size 1151 bp), only single band is observed. The band is located between 1000-1200 bp marker at lane 1, which is consistent to the gBlock original size. Upon restriction with NdeI, the expected bands are 271 bp and 880 bp in size, while restriction with SalI will yield expected bands of 249 bp and 902 bp. At lane 3 and 4, there are three bands observed, indicating that the gBlocks were partially digested with respective enzymes, NdeI and SalI. The heaviest bands in lane 3 and 4 were parallel with the band in lane 2, indicating that the bands were uncut gBlocks. The middle bands in lane 3 and 4 were similar in size (around 900 bp in size), representing the larger fragments of digestion products with their respective enzymes. The lightest bands in lane 3 and 4 were also similar in size, located between 200-300 bp marker at lane 1. They represent the smaller fragments of digestion products. The smaller fragment upon NdeI digestion is 271 bp, while upon SalI digestion is 249 bp. The lightest band in lane 3 migrated slightly slower than lightest band in lane 4, denoting larger size of smaller fragment in lane 3 than lane 4, which is consistent to the expected restriction results.
Ultraviolet (UV) Check
First, we acquired BBa_K592024 by transforming wild-type Escherichia coli K-12 with plasmid in well 17C of Distribution Kit Plate 1, isolating the plasmid, and performing restriction with EcoRI and PstI restricton enzymes. Our “Improved BFP” and BBa_K592024 were separately cloned into plasmid pQE80L using EcoRI and PstI according to our laboratory protocol. The resultant ligation products were subsequently transformed into wild-type E. coli K-12, along with empty pQE80L as control. Transformation products were spread into Luria-Bertani (LB) agar medium containing ampicillin with ratio 1000:1. On the following day, the fluorescence was assessed qualitatively under ultraviolet (UV) light (Figure 3).
User Reviews
UNIQ566f9c2a8b6cd391-partinfo-00000000-QINU UNIQ566f9c2a8b6cd391-partinfo-00000001-QINU