"
Team:Illinois/Notebook
From 2009.igem.org
(→Phil's Box) |
|||
| Line 49: | Line 49: | ||
== '''September 26''' == | == '''September 26''' == | ||
| - | PCRed sRNA target sequences OmpC, OmpA, OmpF, FtsZ, PtsG, GalK,. For PCRs, | + | PCRed sRNA target sequences OmpC, OmpA, OmpF, FtsZ, PtsG, GalK,. For PCRs, ran a 50 μL reaction using |
*4 μL chromosomal template | *4 μL chromosomal template | ||
*0.4 μL of each primer | *0.4 μL of each primer | ||
| Line 57: | Line 57: | ||
*38.4 μL dH<sub>2</sub>O | *38.4 μL dH<sub>2</sub>O | ||
| - | Also digested OmpC, OmpA, and OmpF target sequences from yesterday's PCR using | + | 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) | *1 μL EcoRI-HF (20,000 U/mL) | ||
*3.5 μL 10x Buffer 4 | *3.5 μL 10x Buffer 4 | ||
*30 μL DNA | *30 μL DNA | ||
*0.35 μL BSA | *0.35 μL BSA | ||
| - | *0.15 μL dH<sub>2</sub>O | + | *0.15 μL dH<sub>2</sub>O |
| + | 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 dH<sub>2</sub>O | ||
Also cultured cells containing plasmid PJU-334 and Biobricks BBa_K091101, BBa_I0500, and BBa_R0040 overnight. | 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 | ||
| + | *dH<sub>2</sub>O to 3 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 dH<sub>2</sub>O | ||
<html> | <html> | ||
</div> | </div> | ||
</html> | </html> | ||
{{IllinoisBottomNav}} | {{IllinoisBottomNav}} | ||
Revision as of 21:29, 19 October 2009
Contents |
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.
Post-Summer Progress
This section includes entries for lab work done after the summer was over.
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 3 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
