Difference between revisions of "Team:UChicago/Notebook"

·Made a list of things to order for the future
·Printed most of the protocols; they are located on the lab bench when needed
·Made LB, LB amp, and YED plates
·Note: LB amp plates did not turn out as well as the others; will reattempt on 6/13/2017
·LABELLING ON PLATES
·One red stripe → LB plate
·One red stripe with one blue stripe → LB Amp plate
·One black stripe → YED plates
·The plates have been left on the bench overnight; they will be put in the fridge tomorrow morning. Jason had cleared out a spot for us in the Glick fridge for our use!

·Redid the LB-amp plates
·Made the Drop-Out plates (-Arg)
·Talked to Shannon about cleaning our glassware
·Labelled our glassware
·The petri dishes are in the fridge (all made within the last two days)

·Learned how to use the Nanodrop
·Made two stocks of 500 mL LB media (1000 mL total)
·Testing optimal DNA concentrations for bacterial transformation:
·Spread six different DNA concentrations of the 126 ng/μl plasmid sample (AJ43) Jason gave us onto LB + amp plates
·Left these plates overnight in a 37℃ incubator
   ·Plate 1 = negative control (no DNA, contains 50 μl EB and 50 μl competent cells)
   ·Plate 2 = 453.6 ng DNA (3.6 μL DNA, 46.4 μL EB, 50 μL CC)
   ·Plate 3 = 315 ng DNA (2.5 μL DNA, 47.5 μL EB, 50 μL CC)
   ·Plate 4 = 239.4 ng DNA (1.9 μL DNA, 48.1 μL EB, 50 μL CC)
   ·Plate 5 = 201.6 ng DNA (1.6 μL DNA, 48.4 μL EB, 50 μL CC)
   ·Plate 6 = 163.8 ng DNA (1.3 μL DNA, 48.7 μL EB, 50 μL CC)
·Took a small sample of pOW1 (in frozen bacteria) from stock in the -80℃ fridge
·Put the pOW1 into 5 mL of LB + amp liquid media (1 μL ampicillin / 1 mL LB)
·Left the sample in the 37℃ shaker overnight to incubate

·Retrieved the AJ43 plates
·Negative control = no colonies
·1 = too many colonies to count
·2 = too many colonies to count
·pOW1 plating concentrations for next week:
   ·1 μL pOW1 + 49 μL EB + 50 μL CC
   ·2.5 μL pOW1 + 47.5 μL EB + 50 μL CC
   ·5 μL pOW1 + 45 μL EB + 50 μL CC

·Transformed the pOW1 DNA into competent cells
·Incubated the transformations for 2 hours in a 37℃ shaker
·Spread the samples on LB + amp plates
·Left the plates overnight in a 37℃incubator

·Observed the plates from the previous transformation
·Retrieved pOW1 plates
·Negative Control
   ·No colonies as expected
·5 ul of plasmid + 45 ul of buffer
   Plate
   ·3 colonies
   ·Picked the colonies and put them in LB media
      ·Left in shaker to grow the bacteria (will perform a mini-prep once incubation is complete)
      ·Made a glycerol stock of pOW1 bacteria

·Mini-prepped the pOW1 bacteria that we incubated overnight in the 37℃ shaker
·Sample 1 = 16.5 ng/μL pOW1
·Sample 2 = 17.2 ng/μL pOW1
·pOW1 is in A44 (-80℃ fridge)
·Incubated (in the 37℃ shaker) a small sample of the pOW1 we retrieved from the fridge

·Added MilliQ water to the qcF, qcR, ArgArsF, ArgArsR primers (ng primer x 10 = # μL of water)
·Mutagenesis PCR’d the pSB1C3 with qcF and qcR primers

·PCR’d the pOW1 with ArgArsF and ArgArsR
·Restriction digest the PCR products with DpNI
·Restriction digest the pSB1C3 with HpaI ·Dephosphorylated the DNA
·When we ran a gel of the digest, we saw that the bands of our gel looked distorted
·We did not put in enough TAE buffer for both gels

·Ran the gel again (PCR pSB1C3 products, DpNI cut pSB1C3mut, HpaI cut pSB1C3mut)
·We can see the PCR products, but it’s blurry
·Can’t see anything else
·Redid the PCR for psB1C3 using qcF and qcR
·Transformed the DpNI-digested psB1C3mut into competent cells
·DpNI-digested remaining psB1C3 samples
·Transformed these samples as well

·No colonies grew from our transformations yesterday
·Realized that this is because we plated them on ampicillin plates, whereas psB1C3mut is actually chloroamphenicol resistant
·Made LB + chloroamphenicol plates, plated transformations of DpNI-cut psB1C3mut into competent cells
·Performed a restriction digest on the PCR product (psB1C3mut) from yesterday using DpNI
·Transformed more DpNI-cut psB1C3mut into competent cells and plated them on LB + chloroamphenicol

·Nothing grew on the plates! :( :(
·Performed another restriction digest on the psB1C3mut w/ DpNI using protocol from the GeneHackers manual and w/ the NEB restriction buffer (instead of Promega)
·Transformed the psB1C3mut cut w/ DpNI into MachI competent cells using Jason’s protocol
·DpNI-cut and transformed the psB1C3mut
·Plated the transformation on LB + CAM plates

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July-----

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August-----

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 <button class="accordion">6/29/2017</button>
 <div class="panel">
-   <p>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.</p>
+   <p>
+&#183;Nothing grew on the plates! :( :(<br>
+&#183;Performed another restriction digest on the psB1C3mut w/ DpNI using protocol from the GeneHackers manual and w/ the NEB restriction buffer (instead of Promega)<br>
+&#183;Transformed the psB1C3mut cut w/ DpNI into MachI competent cells using Jason’s protocol<br>
+&#183;DpNI-cut and transformed the psB1C3mut<br>
+&#183;Plated the transformation on LB + CAM plates<br>
+</p>
 </div>