Team:Illinois/Notebook

Notebook

This page will include periodic entries during our research. We will be documenting the progression of our project, any problems or obstacles encountered, and solutions to problems.

Browse by Lab Team

Click on a group name below to view notebook entries and information pertaining to one of our individual lab teams.

• sRNA Library
• 3'-Acting sRNAs
• Hybrid Promoter
• Modeling

Post-Summer Progress

This section includes entries for lab work done after the summer was over.

September 22

PCRed the plasmids containing the Lac promoter, the Tet promoter, CFP, YFP, and GFP. The gel indicated poor results. We found that the primers for the fluorescent protein genes can anneal on their overhang as well. We will need to order new primers for these genes.

September 24

PCRed all target sequences w/ BB sRNAs w/ no results

September 25

PCRed sRNA genes (MicC, MicA, MicF) and their respective target sequences (OmpC, OmpA, OmpF). The gel was run with the Vogel primers as a control since the previous attempt to PCR Biobricks failed.

Also PCRed plasmid pSB1A2. Used 56°C for annealing temperature instead of 58°C and got better results.

Also restreaked plates of E. coli containing plasmid PJU-334 and Biobricks BBa_K091101, BBa_I0500, and BBa_R0040.

September 26

PCRed sRNA target sequences OmpC, OmpA, OmpF, FtsZ, PtsG, GalK,. For PCRs, ran a 50 μL reaction using

• 4 μL chromosomal template
• 0.4 μL of each primer
• 5 μL 10x buffer
• 0.8 μL Pfu polymerase
• 1 μL dNTP mix
• 38.4 μL dH₂O

Also digested OmpC, OmpA, and OmpF target sequences from yesterday's PCR. The first round of digestions was run for three hours using

• 1 μL EcoRI-HF (20,000 U/mL)
• 3.5 μL 10x Buffer 4
• 30 μL DNA
• 0.35 μL BSA
• 0.15 μL dH₂O

The second round of digestions used

• 0.2 μL PstI (100,000 U/mL)
• 3.5 μL 10x Buffer 3
• 30 μL DNA
• 0.35 μL BSA
• 1 μL dH₂O

Also cultured cells containing plasmid PJU-334 and Biobricks BBa_K091101, BBa_I0500, and BBa_R0040 overnight.

September 27

Miniprepped cells containing plasmid PJU-334 and Biobricks BBa_K091101, BBa_I0500, and BBa_R0040. Also digested target sequences from yesterday. For the first round of digestions, ran for three hours using

• 0.4 μL PstI (100,000 U/mL)
• 3.5 μL 10x Buffer 3
• 30 μL DNA
• 0.35 μL BSA
• dH₂O to 35 μL

For the first round of digestion, the incorrect buffer may have been used. The digestion was then cleaned up by PCR cleanup, and then the second round of digestions were run for 2.5 hours using

• 2 μL EcoRI-HF (20,000 U/mL)
• 4 μL 10x Buffer 4
• 30 μL DNA
• 0.4 μL BSA
• 3.6 μL dH₂O

September 28

PCRed the Lac promoter, the Tet promoter (BBa_R0040), and the Tet promoter-RBS construct in a 100 μL reaction using

• 2 μL template
• 0.8 μL each primer
• 20 μL 5x Phusion HF Buffer
• 2 μL dNTP mix
• 0.6 μL Phusion polymerase
• 73.8 μL dH₂O

Also performed dpnI digestion (3 μL) for 4.75 hours.

September 29

PCRed sRNA genes DicF, gcvB, and SgrS out of the E. coli chromosome in 50 μL reactions using

• 4 μL template
• 0.4 μL each primer
• 5 μL 10x buffer
• 1 μL dNTP mix
• 0.8 μL Pfu polymerase
• 38.4 μL dH₂O

September 30

Digested the Lac promoter, Tet promoter, and Tet promoter-RBS construct from the September 28 PCR for four hours using

• 0.4 μL PstI (100,000 U/mL)
• 30 μL DNA
• 3.5 μL BSA
• 0.5 μL dH₂O

Also ligated and transformed Vogel sRNAs, but did not see any colonies.

October 1

Digested sRNA genes DicF, SgrS, and GcvB for 2:20 hours using

• 0.4 μL PstI (100,000 U/mL)
• 3.5 μL 10x Buffer 3
• 30 μL DNA (29 μL for DicF)
• 0.35 μL BSA
• dH₂O to 3 μL

Performed a PCR cleanup on reaction afterwards.

Also PCRed GFP, CFP, and YFP genes using

• 1 μL template
• 0.4 μL each primer
• 10 μL 5x Phusion HF Buffer
• 1 μL dNTP mix
• 0.3 μL Phusion polymerase
• 36.9 μL dH₂O

October 2

Ligated target sequences of OmpA, OmpC, and OmpF into Vogel plasmids:

Ligated for 1:40 hours, transformed by heat shock by putting cells on ice for 30 minutes, in 42°C bath for 45 seconds, and 1 hour recovery.

October 3

The ligations were unsuccessful. No colonies appeared on the plates.