Difference between revisions of "Team:UChicago/Notebook"
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-PCR amplified arg-ars sequence with 1 min per Kb (5 min since Arg-ars sequence is 4.86 Kb)</br> | -PCR amplified arg-ars sequence with 1 min per Kb (5 min since Arg-ars sequence is 4.86 Kb)</br> | ||
-Labeled in blue marker</br> | -Labeled in blue marker</br> | ||
| + | |||
| + | </br></br> | ||
| + | |||
| + | <b>6/19/18</b> | ||
| + | -PCR amplified arg-ars sequence with 30 seconds per Kb (2.5 minutes since Arg-ars sequence is 4.86 Kb)</br> | ||
| + | -Labeled in black marker</br> | ||
| + | -Gel purification Arg-ars PCR products</br> | ||
| + | -Made 0.8% agarose gel</br> | ||
| + | -0.32 g agarose; 40 mL 1X SB buffer; 1µL EtBr </br> | ||
| + | -Each well has 25µL of sample</br> | ||
| + | -Well 1 loaded with 1 min per Kb arg-ars PCR product</br> | ||
| + | -Well 3 loaded with DNA ladder---</br> | ||
| + | -Well 5 loaded with 30 sec per Kb arg-ars PCR product</br> | ||
| + | -Ran at 120V for 25 min (4.86 Kb sequence)</br> | ||
| + | -Well 5 came out clear, however wells 1 and 3 were diffuse to be observed</br> | ||
| + | -Suspected problem: holding micropipette to the bottom of the well when pushing to the second stop, when pulling the micropipette out, dye doesn’t remain | ||
Revision as of 03:22, 14 October 2018
Notebook
6/14/18 -Got familiar with lab -Made LB and normal YPAUD liquid media -Labelled liquid media and left on bench shelf -Make chloramphenicol plates -250mg chloramphenicol; marked by CAM 6/15/18 -Made Arg- YPAUD liquid media (-Used CSM -Arg, -Ura; added uracil in a 1:1 ratio with CSM (1.7g of CSM -Arg -Ura, 1.7g of uracil) -Marked by 3 blue stripes -Made Arg- YPAUD plates -Used CSM -Arg, -Ura; added uracil in a 1:1 ratio with CSM -Marked by three blue stripes -PCR amplify arg-ars sequence -Total volume: 50µL -35µL water -10µL 5X phusion buffer -1µL dNTPs -2.5µL 10µM primer stock (forward and reverse arg-ars primers) -1µL pOW1 (17.6 ng/µL) DNA stock (0.8µL DNA, 4.2µL H2O) -0.5µL phusion -Stored in refrigerator next to PCR machine (labeled iGEM arg-ars PCR) 6/18/18 -Gel electrophoresis of arg-ars sequence (PCR amplified on 6/15/18) -Check if PCR was successful -Agarose gel: -40 mL 1X SB buffer, 0.32g agarose -Mixed and microwaved for 35 seconds -Added 2µL EtBr to agarose gel in flask -Agarose gel opaque and gray when fully polymerized -Sat in chamber, completely covered in buffer, until ready to run the gel -Gel was unsuccessful, neither the ladder band nor the PCR product band could be observed -Suspected problem: samples not loaded into wells properly -Large smear of DNA across wells observed -PCR amplified arg-ars sequence with 1 min per Kb (5 min since Arg-ars sequence is 4.86 Kb) -Labeled in blue marker 6/19/18 -PCR amplified arg-ars sequence with 30 seconds per Kb (2.5 minutes since Arg-ars sequence is 4.86 Kb) -Labeled in black marker -Gel purification Arg-ars PCR products -Made 0.8% agarose gel -0.32 g agarose; 40 mL 1X SB buffer; 1µL EtBr -Each well has 25µL of sample -Well 1 loaded with 1 min per Kb arg-ars PCR product -Well 3 loaded with DNA ladder--- -Well 5 loaded with 30 sec per Kb arg-ars PCR product -Ran at 120V for 25 min (4.86 Kb sequence) -Well 5 came out clear, however wells 1 and 3 were diffuse to be observed -Suspected problem: holding micropipette to the bottom of the well when pushing to the second stop, when pulling the micropipette out, dye doesn’t remain
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Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat.
Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat.