Line 97: | Line 97: | ||
</br></br> | </br></br> | ||
− | <b>6/15/18</b | + | <b>6/15/18</b></br> |
-Made Arg- YPAUD liquid media </br> | -Made Arg- YPAUD liquid media </br> | ||
(-Used CSM -Arg, -Ura; added uracil in a 1:1 ratio with CSM (1.7g of CSM -Arg -Ura, 1.7g of uracil)</br> | (-Used CSM -Arg, -Ura; added uracil in a 1:1 ratio with CSM (1.7g of CSM -Arg -Ura, 1.7g of uracil)</br> | ||
Line 114: | Line 114: | ||
-Stored in refrigerator next to PCR machine (labeled iGEM arg-ars PCR)</br> | -Stored in refrigerator next to PCR machine (labeled iGEM arg-ars PCR)</br> | ||
+ | </br></br> | ||
+ | |||
+ | <b>6/18/18</b></br> | ||
+ | -Gel electrophoresis of arg-ars sequence (PCR amplified on 6/15/18)</br> | ||
+ | -Check if PCR was successful </br> | ||
+ | -Agarose gel: </br> | ||
+ | -40 mL 1X SB buffer, 0.32g agarose</br> | ||
+ | -Mixed and microwaved for 35 seconds</br> | ||
+ | -Added 2µL EtBr to agarose gel in flask</br> | ||
+ | -Agarose gel opaque and gray when fully polymerized </br> | ||
+ | -Sat in chamber, completely covered in buffer, until ready to run the gel </br> | ||
+ | -Gel was unsuccessful, neither the ladder band nor the PCR product band could be observed</br> | ||
+ | -Suspected problem: samples not loaded into wells properly</br> | ||
+ | -Large smear of DNA across wells observed</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> | ||
Revision as of 03:20, 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
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