IGEM:MIT/2006/Notebook/2006-6-27

New BAMT Primer

5' - GTT TCT TCG AAT TCG CGG CCG CTT CTA GAT GAA GAA ACT TTT GTG TAT G - 3'

Results from Smell Experiment

See 6/23/06's Notebook for protocol http://openwetware.org/index.php?title=IGEM:MIT/2006/Notebook/2006-6-23

Cells put in 37 degree Celsius room at 12:00 PM

OD600 Readings (4:30 PM Reading/8:30 PM Reading/10:00 AM Reading)

LBK SAMT 4 Hr Induction (Mistake): 0.33/1.23/2.78 LBK SAMT 4 Hr Induction: 0.56/1.22/2.70 LBK SAMT 8 Hr Induction: 0.55/1.26/3.10

LBK BSMT Control: 0.28/1.07/2.34 LBK BSMT 4 Hr Induction: 0.67/1.47/2.84 LBK BSMT 8 Hr Induction: 0.29/1.05/2.62

EZK SAMT Control: 0/0.16/1.45 EZK SAMT 8 Hr Induction: 0/0.17/0.37 EZK SAMT 8 Hr Induction: 0/0.22/0.48

EZK BSMT Control: 0/0/0.03 EZK BSMT 8 Hr Induction: 0/0/0 EZK BSMT 8 Hr Induction: 0/0.01/0.01

Smell Results

Barry, Stephen, and Andre all agreed with the following conclusions:

The 4 Hr Inductions smelled better than the 8 Hr Inductions for the LBK cultures.

SAMT smelled better in general than BSMT.

BSMT did not smell in EZK (cells did not grow).

The SAMT EZK cultures smelled much less pleasant than the SAMT LBK cultures and the BSMT LBK cultures. There was a weak wintergreen smell in the SAMT EZK cultures but not a very strong smell.

Bad Note: A few of us thought that the LBK BSMT Control smelled minty. Maybe our senses of smell are skewed at this point.

Restriction Digest

1. We are cutting the backbone and the BSMT pcr product separately (using EcoRI and PstI)
2. Total reaction volume is 50μL
3. Our control cut reaction is with the part 3k3.I7101 from Barry
4. the total volume desired is about 50 μL and amounts of dna desired is about 800 nanograms (it was 400ng last time, but Samantha said 1mg so we doubled.
   • pSBIAK3 backbone at 20 ng/μL -----add 28 μL backbone (because we thought it was 30ng/μL before)
   • BSMT pcr product at 90 ng/μL -----add 9 μL
   • control part 3k3.I7101 at 44 ng/μL ----add 20 μL
5. we are also going to use Dpn1 to destroy all dna that is not a pcr product (i.e. get rid of extra template)
6. BACKBONE cut reaction (in order added, following the Knight Lab restriction diigest protocol):
   • 15 μL water
   • 5 μL NEB buffer 2
   • .5 μL BSA
   • 28 μL backbone plasmid
   • .5 μL Pst1
   • .5 μL EcoR1
   • .5 μL Dpn 1
7. BSMT pcr cleanup product cut reaction (in order added, following the Knight Lab restriction diigest protocol):
   • 34 μL water
   • 5 μL NEB buffer 2
   • .5 μL BSA
   • 9 μL BSMT pcr cleanup product
   • .5 μL Pst1
   • .5 μL EcoR1
   • .5 μL Dpn 1
8. CONTROL (Barry's biobrick) cut reaction (in order added, following the Knight Lab restriction digest protocol):
   • 23 μL water
   • 5 μL NEB buffer 2
   • .5 μL BSA
   • 20 μL part 3k3.I7101
   • .5 μL Pst1
   • .5 μL EcoR1
   • .5 μL Dpn 1
9. Incubate both tubes in 37c room for 4-6 hours, then 20 mins at 80deg C to heat inactivate the enzyme, then 4deg forever.