Monday 5/20/13

        Today was basically spent preparing solutions and plates to be used the following day in the competent cell protocol.  In the interest of time, we used a commercial SOB mixture instead of the iGEM formula.  LB agar was used as the growth medium for our plates and one batch was treated with 1 mL of 500x ampicillin.  We had problems with solubility making a 1000x solution with ampicillin trihydrate, so instead used a 500x solution of ampicillin salts.  1 mL aliquots of ampicillin were stored for later use.  Lastly, we streaked both TOP10 and DH5α cells on LB plates and incubated overnight  to be used in the competent cell protocol the following day.

• Prep (2x) 250 mL batch of SOB medium (Used prepared SOB mixture)

• SOB - 250 mL batch

• Yeast - 1.25 g
• Tryptone - 5 g
• NaCl - 0.146 g
• KCl - 0.0465 g
• MgSO4 - 0.6 g

• Autoclave mixture
• Store @ R.T.

• Prep 100 mL batch of SOB medium (Used prepared SOB mixture)

• SOB - 100 mL batch

• Yeast - 0.5 g
• Tryptone - 2 g
• NaCl - 0.0584 g
• KCl - 0.0186 g
• MgSO4 - 0.24 g

• Autoclave mixture
• Store @ R.T.

• Prep 100 mL batch of SOC medium (Used prepared SOB mixture)

• SOC - 100 mL batch

• 100 mL SOB mixture
• 20 mM glucose (not added until 5/21)

• Autoclave mixture
• Store @ R.T.

• Prep (20x) LB plates (Pour thin film into petri dishes)

• LB Agar - 500 mL batch

• LB Agar - 20 g
• Autoclave mixture

• Let set up overnight @ R.T. (no marks)

• Prep (20x) LB plates w/ ampicillin (Pour thin film into petri dishes)

• LB Agar - 500 mL batch

• LB Agar - 20 g
• Autoclave mixture
• 500x ampicillin solution - 1 mL (add after mixture cools somewhat)
• 500 mg ampicillin / 10 mL solution (use DI water)
• Store 1 mL aliquots in -20° C freezer

                        (Use ampicillin salt, not trihydrate)

• Let set up overnight @ R.T. (w/ black marks)

• Prep seed stocks

• Streak 1x - TOP10 & 1x - DH5α cells on LB plates
• Grow overnight @ 37° C

Tuesday 5/21/13

        Today we continued preparation for the competent cells protocol, and decided to use Alex’s protocol over the iGEM protocol.  We prepared magnesium chloride and calcium chloride / glycerol solutions and stored them in the cold locker for the next day.  We also picked colonies from our both of our seed stocks and grew them up overnight in the shaker.  Lastly, we filtered glucose into one of our 100 mL SOB stocks to finish the preparation of the SOC stock.

• Prep 100 mM MgCl2 solution

• 500 mL batch

• 10.15 g MgCl2 ∙ 6H2O (MW - 203.3)

• Autoclave mixture
• Store @ ~0° C

• Prep 100 mM CaCl2 - 15% glycerol solution

• 100 mL batch

• g CaCl2 ∙ 2H2O (MW - 147.0) (Up to 85 mL)
• 15 mL glycerol

• Autoclave mixture
• Store @ ~0° C

• Pick colony and grow in 5 mL SOB overnight in shaker @ 37° C

• 1x - TOP10
• 1x - DH5α

• Filter glucose solution for SOC stock

• 0.36 g glucose (20 mM)
• Dissolve in SOB

Wednesday 5/22/13

        Today we finished the competent cell protocol and also transformed the plasmids for 5 promoters and one RFP construct.  We stored cell stocks for both the TOP10 and DH5α cells, but only the TOP10 cells were used to make the competent cells.  In addition, we prepared chloramphenicol plates for later use.  We once again had solubility issues with our antibiotic, and found that 100% ethanol should be used as the solvent to make a 1000x solution.

• Prep cell stock (2x) from overnight cultures

• 400 μL glyerol
• 600 μL cells (1x - TOP10, 1x - DH5α)
• Store @ -70° C

        (2nd shelf from bottom, rightmost box in freezer)

• Prep competent cells

• 1.2 mL cells in 250 mL SOB (2x - TOP10)
• Place in shaker @ 37° C
• Take periodic absorbance measurements (OD - 0.4-0.6)

(2 samples, one denoted with “*”)

• Centrifuge and resuspend cells (2x)

• Spin down at 5000 rpm for ~10 min. @ 4° C
• Remove supernatant and resuspend with 50 mL MgCl2
• Keep on ice for ~20-30 min.
• Spin down at 4000 rpm for ~10 min. @ 4° C
• Remove supernatant and resuspend with 5 mL CaCl2 - 15% glycerol
• Store 170 μL aliquots @ -70° C

• Transform biobricks into competent cells (I13507,R0011,J23100,J23108,J23110,J23114)

• Add 1 μL DNA for each promoter into 170 μL aliquot of competent cells
• Incubate on ice for ~5 min.
• Heat shock - place in heat bath @ 37° C for ~10 min.
• Add 1 mL SOC and place in shaker for ~1 hr.
• Spread transformed cells on LB ampicillin plates and store overnight @ 37° C

                        (Used 1+ mL, but should have only used ~200 μL)

• Prep (20x) LB plates w/ chloramphenicol (Pour thin film into petri dishes)

• LB Agar - 500 mL batch

• LB Agar - 20 g
• Autoclave mixture
• 1000x chloramphenicol solution - 0.5 mL
• 250 mg chloramphenicol / 10 mL solution (use 100% ethanol)
• Store 0.5 mL aliquots in -20° C freezer

                (Use ethanol as solvent, water does not work)

• Let set up overnight @ R.T. (w/ red marks)

Thursday 5/23/13

        Unfortunately only 2 of our 6 transformations produced cell cultures, so today we designed some experiments to test several factors in our transformation protocol.  The two that did work produced a very low yield.  J.F. mini prepped the colonies that did form, and Isra worked on an electroporation technique to try and produce cell cultures for the 4 failed transformations.  Bill, Rayleigh and I worked on the transformation experiments.

        Experiment #1:  The suspected issues with our transformation procedure were the concentration of genetic material and the method of heat shock.  In the 1st trial, we wanted to test to make sure the the competent cells would grow on an agar plate.  For the 2nd trial, we tested the original protocol using commercial pUC19 plasmids to ensure that the genetic material was viable.  For trials 3-5, we increased the concentration of pUC19 and varied the method of heat shock to allow us to determine which method produced cells with the highest efficiency.  We did 2 samples for each trial to demonstrate repeatability.  This was still not a completely controlled experiment however.  We deviated from the original protocol by increasing the on ice incubation period from ~5 to ~30 min. and also increased the recovery time from ~1 hr. to ~1.5-2 hrs. in the shaker.  In addition, I made the mistake of plating 1 mL of the competent cells / growth medium yesterday when only 200 μL at most was necessary.  This time we plated ~200 μL.  Details of the experimental setup are given below.

• Only 2 out of 6 plates produce cultures (J23100, I13507), those had a low yield
• Experiments:

• Thaw competent cells on ice for ~15 min.
• Separate into 50 mL aliquots
• Add pUC19 (if necessary) - (pUC19 - 10 pg / μL)
• Incubate on ice for ~30 min.
• Heat shock in water bath (if necessary)
• Add 1 mL SOC medium
• Shake @ 37° C for 1+ hr. (note: 2 hr. recommended by iGEM protocol)
• Plate 200 μL, store excess @ 4° C
• Put plates in incubator @ 37° C overnight

• Store all unused prepared plates @ 4° C
• Put plates with original transformation back in incubator @ 37° C for another night.

Friday 5/24/13

        Today we evaluated the results of our experiments and began preparing for next week.  One of the transformations that we put in for a 2nd night produced a few cell colonies.  We also found that our SOC stock had been contaminated at some point on thursday.  Our experiment showed that the competent cells we had prepared were viable, but not very efficient.  We decided to prepare a new batch of competent cells using the iGEM protocol, and prepared several batches of SOB using the iGEM formula (also found on wikipedia).  We also made some more ampicillin plates.  Two of our experimental trials (4 and 5) for the transformation were inconclusive.  Our best and most reliable results were produced using 50 μL cells / 2 μL pUC19 and heat shocking for ~10 min. @ 37° C.  We will adjust our transformation protocol accordingly, and also make changes to allow for ~30 min. incubation on ice prior to heat shock and ~2 hr recovery following the heat shock.

• Plate with J23108 grew cultures overnight (low yield)
• SOC stock had been contaminated

• Prep (2x) 250 mL batch of SOB medium (used iGEM formula)

• Details of formula found above (Mon. 5/20/13)

• Prep 100 mL batch of SOB medium (Used prepared SOB mixture)

• Details of formula found above (Mon. 5/20/13)

• Prep 100 mL batch of SOC medium (Used prepared SOB mixture)

• Details of formula found above (Mon. 5/20/13)
• Glucose will not be added until Monday 5/27

• Prep (20x) LB plates w/ ampicillin

• Details of protocol found above (Mon. 5/20/13)
• 1 mL ampicillin 500x aliquot retrieved from -20° C freezer

• Let set up overnight @ R.T. (w/ black marks)

• Experimental results: