Team:TP CC-SanDiego/Notebook

NOTEBOOK
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WEEK 1

Day 1 afternoon: August 3, 2015: (Nathan, Aidan, Abhi)


SUMMARY:

  • Transformation of E. coli Dh5-alpha with ordered plasmid.


WHAT TO DO NEXT:

  • Culture GOOD bacterial colonies

  • Replate colonies on LB+Amp Agar plates and incubate at 37deg C

  • Create 2-3 new plates of transformed E. coli, following the same procedure as above - aliquot 5uL of the plasmid to prevent contamination of the "master broth"



Day 2 morning: August 4, 2015: (Caroline, Patricia, Dane, Abhi)


WHAT WE FOUND:

  • No colonies on either plate-> put both plates back in the incubator


SUMMARY:

  • Transformation of E. coli Dh5-alpha with ordered plasmid. (we used 2.5 ul diluted plasmid as opposed to the 1 ul used yesterday)

  • Made changes to transformation protocol-> centrifuged to concentrate bacteria


Day 2 afternoon: August 4, 2015: (Nathan)


SUMMARY:

  • Checked on plates



Day 3 afternoon: August 5, 2015: (Nathan, Anthony, Catherine, Madeline)


WHAT WE FOUND:

  • Colonies grew on both plates from August 4; plates from August 3 still had no colonies


SUMMARY:

  • Streak plated colonies from the more populated of the two original plates onto two more

  • Created 6 LB+Amp plates


WHAT TO DO NEXT:

  • Nathan - autoclave remaining LB+Agar to create additional plates because LB+Amp agar solidified; make more plates

  • IPTG induction and SDS Page



Day 4 morning: August 6, 2015: (Caroline, Patricia)


SUMMARY:


PROCEDURE:

  • Calculated Protein Size (LbCHI31) by entering amino acid sequence in http://web.expasy.org/compute_pi/ : 33 kDa

  • Made a Stacking Gel solution and Separating Gel solution for 12% Gel (we added everything except APS & TEMED which should be in the 4C fridge)


WHAT TO DO NEXT:

  • Finish making gels (instruction sheet in iGEM box): solutions are in 15mL falcon tubes on the iGEM 4C fridge rack

  • Run SDS Page

  • Make Stock solutions(if you have time) for 10% SDS, 1.5 M Tris(ph=8.8), and 0.5M Tris-HCl(ph=6.8) so that we don’t have to keep using other people’s



Day 4 afternoon: August 6, 2015: (Aidan, Catherine, Patricia, Anthony, Jonathan)


SUMMARY:

  • We made 2 12% page gels


WHAT TO DO NEXT:

  • Make more stock solutions for future use (maybe 1.5M Tris ph 8.8, 1.0M Tris ph 6.8, 10% SDS, TAE buffer)

  • http://openwetware.org/wiki/Qiagen_Buffers We should make sure we have tools necessary to conduct minipreps

  • Basically stock up on things we will be needing in the future to make more gels, run said gels, conduct miniprep, western blots

  • Next week Monday we will likely make an overnight saturated culture of our E.Coli and run a miniprep (if we can) on Tuesday



Day 5 morning: August 7, 2015: (Patricia, Caroline, Catherine) Time: 09:30 - 12:00


SUMMARY:

  • We made 10% SDS Solution, 1.0M Tris pH 6.8, and 1.5m Tris pH 8.8

  • All three solutions are stored at room temperature.


WHAT TO DO NEXT:

-Create TAE Buffer.



Day 5 afternoon: August 7, 2015: (Aidan, Madeline, Priya ) 2:35-3:30


SUMMARY:

  • Made 200mL LB broth for future use and saturated culture.


WHAT TO DO NEXT:

  • Probably remake a saturated overnight culture (LB + e.Coli + incubate overnight)

  • Make some TAE buffer (if we need to)




WEEK 2

Day 6 morning: August 10, 2015: (Nathan, Caroline, Abhi, Patricia): 10:00-12:30


SUMMARY:

  • Made 500 mL TAE buffer for SDS Page (on iGEM shelf)

  • Created an overnight (4-6 hour) culture for SDS Page (in top 37 deg.C shaker)


WHAT TO DO NEXT:

  • Perform SDS Page (Save analysis for tomorrow) - Store in fridge after de-dyeing

    • Store by submerging in ddH2O (use gel container - ask Aaron/Bryan/Reta)



Day 6 afternoon: August 10, 2015: (Anthony, Liana, Nathan, Dane, Edric)


SUMMARY:

  • Ran a gel for SDS Page analysis (Ladder, control, and three copies of the samples)

  • Staining gel in Coomassie Blue overnight


WHAT TO DO NEXT: (Just making notes here for the rest of this week- Nathan)

  • Analyze SDS page results and see if procedure worked well (Monday afternoon = test run) - Tuesday morning + afternoon

  • Make one set of stacking gels for an SDS run to save time on Wednesday (if possible) - Tuesday afternoon

  • Two sets of stacking gels for two more runs of SDS page - Wednesday afternoon

  • Prepare LB+Amp+colony vs LB+e.coli for SDS page runs - Thursday morning

  • Run both gels at the same time in SDS gel box - Thursday afternoon

  • Analyze gel results (expression) - Friday morning and afternoon



Day 7 morning: August 11, 2015 9:00-10:30 (Patricia, Priya, Gita)


SUMMARY:

  • Prepared solutions for 2 12% stacking gels


WHAT TO DO NEXT:

  • Finish making Gels



Day 7 morning: August 11, 2015 11:30-1:30 (Caroline)


SUMMARY:

  • Staining and destaining gel


WHAT TO DO NEXT:

Gel Analysis:

  • Scan gel

Create 2 more gels:

  • Solutions w/o AP and TEMED are on the rack in 15 ml tubes

  • Gel rig is on bench with only one set of gels

  • Follow procedure from Day 4 and finish gels



Day 7 afternoon: August 11, 2015 (Nathan, Caroline, Liana, Dane, Abhi)


SUMMARY:

  • Made 2 12% stacking gels (remade gel solutions from morning)

  • Stained and destained gel from Monday (used new Coomassie instead of used one)

  • Performed Gel Analysis -> no results


WHAT TO DO NEXT:

Morning:

  • Do Transformation and grow on 2 new LB+Amp plates → use Day 2 morning procedure for this

  • Create new culture of LB+sample -> 3 samples

  • IMPORTANT: REHYDRATE OLD GEL


Afternoon:

  • Run two new gels for SDS page -> IMPORTANT: Load the ladder, the 3 samples that Caroline and Nathan made, and the 3 samples that Liana and Nathan made

  • Stain and destain gels; scan; save → we can analyze through discussions later


Notes:

  • Run old SDS gel → use old plates as positive control for cells on new plates



Day 8 morning: August 12, 2015 (Nathan, Liana)


SUMMARY:

  • Made O/N (4-6 hour) LB+Amp+culture for SDS page


WHAT TO DO NEXT:

  • Check old gel for dehydration - if not dehydrated, leave in wrap… if dehydrated, rehydrate

  • Run the two gels made on 8/11/15… Refer to 8/11/15 on notes regarding loading the gel (remember to keep the two end lanes open)

  • Aidan and Anthony run the gels

  • Madeline, Priya, Gita create two new plates (in 4 degC fridge)



Day 8 afternoon: August 12, 2015 (Aidan, Madeline, Dane, Priya, Gita)


SUMMARY:

  • Made 3 O/N saturated cultures and 1 control (in 37 degC shaker)



Day 9 morning: August 13, 2015 (Nathan, Caroline, Madeline, Catherine)


SUMMARY:

  • Ran an SDS Page gel (1 ladder, 3 samples, 3 buffer+SDS running dye (ensures that everything runs straight))

  • Made another saturated batch of LB+Amp+colony.


WHAT TO DO NEXT:

  • Stain and destain gel

  • Scan gel and upload with the light box

  • Use fluorescence to look for our GFP tag around 33 kDA (size of LbCHI31)


NOTES:

  • The reason we made another batch is because our plasmid, pUCIDT, is a custom plasmid off IDT that was selected by IDT to meet our lab requirements. Unfortunately, pUCIDT does not contain a lac operon (so no IPTG induction), is a DNA specific plasmid (so it doesn’t over express proteins, but it’ll still express just at a lower concentration, which is why we created a new batch with 6 mL of LB for greater growth. The logic behind this is, because we can’t use IPTG induction, we will detect the expression of our plasmid at 33 kDa via the GFP tag we linked to our protein), and does not have common restriction enzyme cut sites (eg. ECOR1, XBal, etc).



Day 9 evening: August 13, 2015 (Aidan, Anthony, Patricia)


SUMMARY:

  • Destained SDS Page gel from earlier today; left overnight on shaker in diH2O (step 11)

  • Set-up saturated cultures for SDS Page tomorrow evening


WHAT TO DO NEXT:

  • Gel from today’s SDS Page was left overnight on the dark room shaker in diH2O. Laminate the gel (use the binder plastic page covers in the drawers of the dark room in front of the shaker) and analyze.



Day 10 morning: 8/14/15 (Caroline, Madeleine, Jonathan, Cat*)


SUMMARY:

  • Checked on gel

  • Spectrophotometry: measured Concentrations



Day 10 afternoon: 8/14/15 (Aidan Catherine, Anthony)


SUMMARY:

  • We ran SDS Page with a ladder, control (wt Dh5-alpha E. coli), and 2 samples (sample 1 had no visible pellet [not enough growth] ) in that order

  • Left said gel in coomassie blue stain O/N


WHAT TO DO NEXT:

  • Destain gel and scan both gels (other one was from Thursday) (both on shaker)

  • Analyze gels

  • Look at LB + Amp plates under UV to see GFP + take pictures




WEEK 3

Day 11 morning: 8/17/15 (Nathan, Caroline)


SUMMARY:

  • Destained gel from 8/14/15

  • Scanned gel from 8/13/15


WHAT TO DO NEXT:

  • Leaving the gel in Coomassie Blue stained gel really heavily, so continue to destain (if needed)

  • Look at LB + Amp plates and check for GFP

  • Store gels in box (submerge in diH2O)



Day 11 Evening: 8/17/15 (Anthony)


SUMMARY:

  • Finished destaining 8/14/15 gel and scanned gel

  • Setup for saturated samples for SDS Page tomorrow (redo)


WHAT TO DO NEXT

  • MORNING CREW → Make an SDS Page gel for SDS Page tonight.

  • Autoclave a new stock of LB media

  • AFTERNOON CREW → Run SDS Page with new saturated samples (cultured in sterile LB media from Bryan)

  • Reanalysis and storage for Western Blot → Ask Bryan about drying gel before scanning so that gel can be restored afterwards for further tests.



Day 12 Morning: August 18, 2015 9:00-11:00 (Patricia, Priya)


SUMMARY:

  • Prepared solutions for 2 12% stacking gels

WHAT TO DO NEXT:

  • Finish making gels



Day 13 afternoon: August 19, 2015 3:00 - (Aidan, Madeline)


SUMMARY:

  • Finished making 12% SDS gels (1 gel was given to Norman as he needed to use one)

  • Measured concentration of growth using spectrophotometer

  • Ran SDS Page Gel with Ladder, Control, Sample 1, and Sample 2


WHAT TO DO NEXT:

  • Destain Gels and take already destained gels out of their gel boxes.



Day 14 Morning: August 20, 2015 (Nathan, Caroline)


SUMMARY:

  • Destained gel


WHAT TO DO NEXT:

  • Scan gel



Day 14 Afternoon: August 20, 2015 5:00 - Patricia


SUMMARY:

  • Gel Scan






WEEK 4

Day 15 Evening: August 27th, 2015 (Anthony and Patricia)


SUMMARY:

  • PCR of the chitinase gene




WEEK 5

Day 16 Evening: September 1, 2015 (Nathan and Madeline)


SUMMARY:

  • Made LB+Spectinomycin Agar Plates

  • PCR of chitinase -> Re-did this because PCR product was not in 4 degC or -20 degC



Day 17 Evening: September 3rd, 2015 (Caroline)


SUMMARY:

  • Measured DNA concentrations with spectrophotometer and Nanodrop Tube #1: 45.58 ng/uL Tube #2: 46 ng/uL


WHAT TO DO NEXT:

  • Gibson Assembly (~50 min.)

  • Transformation




WEEK 6

Day 18 Evening: September 11th, 2015 (Caroline)


SUMMARY:

  • red pcr tube in -20C on igem rack contains product


WHAT TO DO NEXT:

  • Transformation into BL21 and Dh5Alpha competent cells

  • Make gels




WEEK 7

Day 19 Morning: September 14, 2015 (Nathan, Anthony, Abhi)


SUMMARY:

  • Made stock solution of LB

  • Transformed 2 BL21 cells and 1 Dh5-Alpha cell to make 2 plates of transformed BL21, 2 plates of transformed Dh5-Alpha, and 1 plate of BL21 negative control

  • Made 12% SDS Page Gels


WHAT TO DO NEXT:

  • Create O/N cultures (2 BL21, 2 Dh5Alpha, 1 negative control, 1 positive control, 1 wild-type BL21, and 1 wild-type Dh5Alpha)

  • Perform Colony PCR



Day 20: September 15th, 2015 (Caroline)


SUMMARY:

  • Checked Plates (7) for Colonies -> left them in 37 deg. incubator

  • Colony PCR

  • Labeled 7 plastic tubes for O/N culture


WHAT TO DO NEXT:

  • Run an agarose gel with the PCR products to see if the bacteria have successfully taken up the ligated plasmid

  • Create saturated colonies (use the tubes already labeled)

  • Run SDS Page Gel