Growing E.Coli
1. 70% ethanol was used to clean the work area.
2. The Amp/Kan petri dishes were labeled Part A and Part B.
3. An inoculating loop was used to remove cells from the Part A slab to the Part A plate, streaking them in a zig-zag pattern.
4. Another inoculating loop was used to create an overlapping zig-zag pattern.
5. Steps 3 and 4 were repeated using Part B.
6. The petri dishes were Parafilmed, inverted, and placed into an 37ºC incubator for 15 hours.
7. The plates were stored at 4ºC until they were needed for cell culture.
2. The Amp/Kan petri dishes were labeled Part A and Part B.
3. An inoculating loop was used to remove cells from the Part A slab to the Part A plate, streaking them in a zig-zag pattern.
4. Another inoculating loop was used to create an overlapping zig-zag pattern.
5. Steps 3 and 4 were repeated using Part B.
6. The petri dishes were Parafilmed, inverted, and placed into an 37ºC incubator for 15 hours.
7. The plates were stored at 4ºC until they were needed for cell culture.
Growing cell cultures
1. The work area was cleaned with 70% ethanol.
2. Two 14 ml were labeled Part A and Part B.
3. 5 ml of Amp/Kan LB Broth were added to each tube.
4. The plates were removed from the fridge.
5. An inoculating loop was used to transfer a colony from the Part A plate to the Part A tube. The tube was then lightly capped.
6. Another inoculating loop was used to transfer a bacteria colony from the Part B plate to the Part B tube. The tube was then lightly capped.
7. The tubes were incubated at 37ºC in a rotator for 16 hours.
8. After 16 hours, the tubes were closed completely and then stored in a 4ºC fridge until needed.
2. Two 14 ml were labeled Part A and Part B.
3. 5 ml of Amp/Kan LB Broth were added to each tube.
4. The plates were removed from the fridge.
5. An inoculating loop was used to transfer a colony from the Part A plate to the Part A tube. The tube was then lightly capped.
6. Another inoculating loop was used to transfer a bacteria colony from the Part B plate to the Part B tube. The tube was then lightly capped.
7. The tubes were incubated at 37ºC in a rotator for 16 hours.
8. After 16 hours, the tubes were closed completely and then stored in a 4ºC fridge until needed.
miniprep
1. 70% ethanol was used to clean the work area.
2. The culture tubes were spun in a centrifuge for 3 minutes at 8000 rpm.
3. The supernatant was poured in a beaker, bleached, and disposed of.
4. 250 ul of Buffer P1 was added to each tube.
5. A pipet was used to re-suspend the cell pellet.
6. The resuspended cells were transferred to a microcentrifuge tube labeled with the name of the part.
7. 250 ul of Buffer P2 was pipetted into each microcentrifuge tube.
8. The tubes were closed and then inverted 5 times.
9. 350 ul of Buffer N3 was pipetted into each tube.
10. The tubes were closed and inverted 5 times.
11. The tubed were centrifuged at 13000 rpm for 10 minutes.
12. The supernatant was pipetted to the appropriately labeled spin column.
13. The spin column tubes were centrifuged at 13000 rpm for 1 minute.
14. The filter tube was removed from the collection tube. The contents of the collection tube were poured into a beaker and the filter tube was placed back into the collection tube.
15. 500 ul of Buffer PB was added to each spin column and steps 13-14 were repeated.
16. 750 ul of Buffer PE was added to each spin column and steps 13-14 were repeated.
17. The samples were spun at 13000 rpm for 1 minute.
18. Each filter tube was transferred to a microcentrifuge tube labeled with the corresponding part name.
19. 50 ul of distilled water was added to each filter tube.
20. The samples were left to sit for 1 minute and then centrifuged for 1 minute at 13000 rpm.
21. The tubes were stored at 4ºC.
2. The culture tubes were spun in a centrifuge for 3 minutes at 8000 rpm.
3. The supernatant was poured in a beaker, bleached, and disposed of.
4. 250 ul of Buffer P1 was added to each tube.
5. A pipet was used to re-suspend the cell pellet.
6. The resuspended cells were transferred to a microcentrifuge tube labeled with the name of the part.
7. 250 ul of Buffer P2 was pipetted into each microcentrifuge tube.
8. The tubes were closed and then inverted 5 times.
9. 350 ul of Buffer N3 was pipetted into each tube.
10. The tubes were closed and inverted 5 times.
11. The tubed were centrifuged at 13000 rpm for 10 minutes.
12. The supernatant was pipetted to the appropriately labeled spin column.
13. The spin column tubes were centrifuged at 13000 rpm for 1 minute.
14. The filter tube was removed from the collection tube. The contents of the collection tube were poured into a beaker and the filter tube was placed back into the collection tube.
15. 500 ul of Buffer PB was added to each spin column and steps 13-14 were repeated.
16. 750 ul of Buffer PE was added to each spin column and steps 13-14 were repeated.
17. The samples were spun at 13000 rpm for 1 minute.
18. Each filter tube was transferred to a microcentrifuge tube labeled with the corresponding part name.
19. 50 ul of distilled water was added to each filter tube.
20. The samples were left to sit for 1 minute and then centrifuged for 1 minute at 13000 rpm.
21. The tubes were stored at 4ºC.
Restriction Digest
1. Lab bench was cleaned with 70% ethanol.
2. Enzymes and buffers were kept in ice.
3. The NEB Buffer 2 and BSA were thawed in room temperature water and then homogenized by inverting and flicking the tubes.
4. Four 0.6 tubes were labeled Part A, Part B, pSB1C3, and RFP Control.
5. 500 ng of DNA and 22.5 ul of distilled water was added to each tube.
6. 5 ul of Buffer 2 was added to each tube.
7. 0.5 ul of BSA was added to each tube.
8. 1 ul of EcoRI enzyme and 1 ul of Spel enzyme was added to Part A tube.
9. 1 ul of Xbal enzyme and 1 ul of Pstl enzyme was added to Part B tube.
10. 1 ul of EcoRI enzyme and 1 ul of Pstl enzyme was added to pSB1C3 tube.
11. 1 ul of EcoRI enzyme and 1 ul of Pstl was added to the RFP Control tube.
12. The contents of each tube were individually pipetted up and down 5 times each and then spun in a microcentrifuge for 5 seconds.
13. The restriction digests were incubated at 37ºC for 30 minutes.
14. The restriction digests were then incubated at 80ºC for 20 minutes.
15. The DNA was stored at 4ºC.
2. Enzymes and buffers were kept in ice.
3. The NEB Buffer 2 and BSA were thawed in room temperature water and then homogenized by inverting and flicking the tubes.
4. Four 0.6 tubes were labeled Part A, Part B, pSB1C3, and RFP Control.
5. 500 ng of DNA and 22.5 ul of distilled water was added to each tube.
6. 5 ul of Buffer 2 was added to each tube.
7. 0.5 ul of BSA was added to each tube.
8. 1 ul of EcoRI enzyme and 1 ul of Spel enzyme was added to Part A tube.
9. 1 ul of Xbal enzyme and 1 ul of Pstl enzyme was added to Part B tube.
10. 1 ul of EcoRI enzyme and 1 ul of Pstl enzyme was added to pSB1C3 tube.
11. 1 ul of EcoRI enzyme and 1 ul of Pstl was added to the RFP Control tube.
12. The contents of each tube were individually pipetted up and down 5 times each and then spun in a microcentrifuge for 5 seconds.
13. The restriction digests were incubated at 37ºC for 30 minutes.
14. The restriction digests were then incubated at 80ºC for 20 minutes.
15. The DNA was stored at 4ºC.
Ligation
1. The lab bench was cleaned using 70% ethanol.
2. One 0.6 ml tube was labeled as New Part.
3. 2 ul from pSB1C3, 3.3 ul from Part A, 3.9 ul from Part B, 1 ul of T4 DNA Ligase Reaction Buffer, and 0.5 ul of T4 DNA Ligase was added to the New Part tube and then pipetted up and down 3x.
4. The New Part tube was microcentrifuged for 5 seconds.
5. Another 0.6 ml tube was labeled as Ligation Control.
6. 2 ul from the RFP Control, 6.5 ul of distilled water, 1 ul of T4 DNA Ligase Reaction Buffer, and 0.5 of T4 DNA Ligase was added to the Ligation Control tube and pipetted up and down 3x.
7. The Ligation Control tube was microcentrifuged for 5 seconds.
8. The tubes were incubated for 30 minutes at 16ºC and then for 20 minutes at 80ºC.
9. The ligated products were stored at -20ºC.
2. One 0.6 ml tube was labeled as New Part.
3. 2 ul from pSB1C3, 3.3 ul from Part A, 3.9 ul from Part B, 1 ul of T4 DNA Ligase Reaction Buffer, and 0.5 ul of T4 DNA Ligase was added to the New Part tube and then pipetted up and down 3x.
4. The New Part tube was microcentrifuged for 5 seconds.
5. Another 0.6 ml tube was labeled as Ligation Control.
6. 2 ul from the RFP Control, 6.5 ul of distilled water, 1 ul of T4 DNA Ligase Reaction Buffer, and 0.5 of T4 DNA Ligase was added to the Ligation Control tube and pipetted up and down 3x.
7. The Ligation Control tube was microcentrifuged for 5 seconds.
8. The tubes were incubated for 30 minutes at 16ºC and then for 20 minutes at 80ºC.
9. The ligated products were stored at -20ºC.
TRANSFORMATION
1. The work area was cleaned with 70% ethanol.
2. All materials were kept on ice.
3. Three 2.0 ml microcentrifuge tubes were labeled Transformation Control, Ligation: New Part, and Ligation Control.
4. 5 ul of RFP Control DNA was added to Transformation Control.
5. 2 ul of New Part ligation product was added to Ligation: New Part.
6. 2 ul of RFP Control ligation product was added to Ligation Control.
7. The tubes were placed on ice.
8. One competent cell aliquot tube was thawed on ice.
9. 50 ul of competent cells were pipetted into each microcentrifuge tube.
10. The DNA was kept on an ice bath for 30 minutes.
11. The tubes were placed in a 42ºC water bath for 60 seconds.
12. The tubes were then placed back on the ice for 5 minutes.
13. 200 ul of Transformation Control was added to the corresponding plate. The plate was shifted around so the liquid covered it completely.
14. Step 13 was repeated for the other plates and their corresponding transformations.
15. The plates were placed into the 37ºC incubator with the agar side facing up for 12-14 hours.
16. The plates were later checked for red colonies.
2. All materials were kept on ice.
3. Three 2.0 ml microcentrifuge tubes were labeled Transformation Control, Ligation: New Part, and Ligation Control.
4. 5 ul of RFP Control DNA was added to Transformation Control.
5. 2 ul of New Part ligation product was added to Ligation: New Part.
6. 2 ul of RFP Control ligation product was added to Ligation Control.
7. The tubes were placed on ice.
8. One competent cell aliquot tube was thawed on ice.
9. 50 ul of competent cells were pipetted into each microcentrifuge tube.
10. The DNA was kept on an ice bath for 30 minutes.
11. The tubes were placed in a 42ºC water bath for 60 seconds.
12. The tubes were then placed back on the ice for 5 minutes.
13. 200 ul of Transformation Control was added to the corresponding plate. The plate was shifted around so the liquid covered it completely.
14. Step 13 was repeated for the other plates and their corresponding transformations.
15. The plates were placed into the 37ºC incubator with the agar side facing up for 12-14 hours.
16. The plates were later checked for red colonies.