IGEM:MIT/2006/Notebook/2006-6-23

To Do

1. Ligation reaction
2. Run gel of everybody PCR
   • Try BAMT and SAMT PCR again with re-diluted primers
3. Dilute (then induce at 4 hrs and 8 hrs) the overnight LB and EZ liquid cell cultures
   • time test for pleasant smells at intervals after IPTG induction
4. Repeat transformation of biobrick ligations into BL21 cells

Induction Smell Experiment

The following tubes were created from 20 uL samples of the overnight BSMT and SAMT cultures:

SAMT + 10 uL LB Kan + 37.20 uL .5 M Salicylic Acid (Not Induced)

SAMT + 10 uL EZ Kan + 37.20 uL .5 M Salicylic Acid (Not Induced)

BSMT + 10 uL LB Kan + 37.20 uL .5 M Salicylic Acid (Not Induced)

BSMT + 10 uL EZ Kan + 37.20 uL .5 M Salicylic Acid (Not Induced)

SAMT + 10 uL LB Kan + 37.20 uL .5 M Salicylic Acid (Induced after 4 hrs)

SAMT + 10 uL EZ Kan + 37.20 uL .5 M Salicylic Acid (Induced after 4 hrs)

BSMT + 10 uL LB Kan + 37.20 uL .5 M Salicylic Acid (Induced after 4 hrs)

BSMT + 10 uL EZ Kan + 37.20 uL .5 M Salicylic Acid (Induced after 4 hrs)

SAMT + 10 uL LB Kan + 37.20 uL .5 M Salicylic Acid (Induced after 8 hrs)

SAMT + 10 uL EZ Kan + 37.20 uL .5 M Salicylic Acid (Induced after 8 hrs)

BSMT + 10 uL LB Kan + 37.20 uL .5 M Salicylic Acid (Induced after 8 hrs)

BSMT + 10 uL EZ Kan + 37.20 uL .5 M Salicylic Acid (Induced after 8 hrs)

Ran Gel

This is the gel from yesterday; the lanes, from left to right, are:

1. ladder
2. SAMT template with SAMT primers
3. BAMT template with BAMT primers
4. BSMT template with BSMT primers
5. SAMT template with T7 & TφR
6. BAMT template with T7 & TφR
7. BSMT template with T7 & TφR
8. ATF1 with yeast genomic DNA template and ATF1 primers

• Judging by the lengths of the bands that were cut by T7, our templates are correct. We think that the primers we used for BAMT and SAMT may have been swtiched...so we will re-do the PCR for BAMT and SAMT, making sure that we use the right primers (we'll go straight from the original container that we got from Invitrogen)

Repeat ligation reaction

1. Remove Backbone/BSMT tube and Backbone/Control part tube from storage in -20c fridge
2. Considerations:
   • want 10 μL total volume
   • want 50 nanograms of both of the desired dnas in each ligation tube: ( i.e. backbone and BSMT -or- backbone and barry's control part)
   • for the spec readings remember that the amount of each desired dna will be HALF of the reading (b/c have 2 dnas in each tube), so make calculations appropriately
3. Ligation BSMT Test:
   • 1 μL T4 DNA ligase buffer (be gentle, also check that it smells like wet dog)
   • .5 μL T4 ligase enzyme
   • 7 μL of BSMT = 50 nanograms of backbone and BSMT pcr product (basically want 100 nanograms of 15.7ng/μL, where 15.7 is from dna spec reading)
   • 1.5 μL water (volume neccesary for 10 μL total volume)
4. Ligation Control Part Test:
   • 1 μL T4 DNA ligase buffer (be gentle, also check that it smells like wet dog)
   • .5 μL T4 ligase enzyme
   • 8 μL control = 50 nanograms of backbone and control part dna (spec = 13 ng/μL)
   • .5 μL water (volume neccesary for 10 μL total volume)
5. Thermocycle for 1.5 hrs at 16c with a 10 minute heat shock at 65c

Repeat transformation

1. See protocol here
2. Considerations:
   • use flat bottom tubes
   • heat shock 50 seconds
   • vortex samples before transforming
3. Plate both 20 μL and 200 μL of BSMT-BB and Control part-BB on Amp/CHL plates
4. store in 37c room for 12-16 hours
   • someone will remove plates early saturday morning to make liquid cultures (assuming we have transformants)

PCR with new primer dilutions (and run a gel)

1. Prepare the following tubes:
   • 1) 1μL "BSMT for pcr" + 49μL of Tom's PCR mix + .6μL of each BSMT primer
   • 2) 1μL "BAMT for pcr" + 49μL of Tom's PCR mix + .6μL of each (new) BAMT primer
   • 3) 1μL "SAMT for pcr" + 49μL of Tom's PCR mix + .6μL of each (new) SAMT primer
     • NOTE: all primers were re-diluted to about 25 μM with careful labels -- and .6 μL yields roughly 300 nM final concentration of each primer in each tube)
2. Run them at: 95deg for 3:00, then cycle through: (a) 94deg for :30 (b) 55deg for :30 (c)68deg for 2:15, then 72deg for 10:00

1. The results of yesterday's gel indicate that the new PCR did NOT work. The BSMT control looked good, but no bands appeared in the SAMT and BAMT lanes.