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Thursday, August 4
From 2011.igem.org
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*The pET-Taq plasmid that was our template for mutagenesis (positive control) | *The pET-Taq plasmid that was our template for mutagenesis (positive control) | ||
| - | ''' | + | '''PCR Recipe''' |
{| border="1" align="center" style="text-align:center;" | {| border="1" align="center" style="text-align:center;" | ||
| - | |Template DNA | + | |Template DNA |
|1 ul | |1 ul | ||
|- | |- | ||
| - | |Primer Pol Taq Fwd prefix | + | |Primer Pol Taq Fwd prefix |
|1 uM , 0.3 ul | |1 uM , 0.3 ul | ||
|- | |- | ||
| - | |Primer Pol Taq Rvs suffix | + | |Primer Pol Taq Rvs suffix |
|1um , 0.3ul | |1um , 0.3ul | ||
|- | |- | ||
| Line 41: | Line 41: | ||
| 31.8 ul | | 31.8 ul | ||
|- | |- | ||
| - | |Total Volume of Rxn | + | |Total Volume of Rxn |
|50 ul | |50 ul | ||
|} | |} | ||
| + | |||
| + | '''Recipe for Mastermix''' | ||
| + | |||
| + | {| border="1" align="center" style="text-align:center;" | ||
| + | |Fwd primer | ||
| + | |15 ul | ||
| + | |- | ||
| + | |Rv primer | ||
| + | |15 ul | ||
| + | |- | ||
| + | |dNTPs | ||
| + | |5 ul | ||
| + | |- | ||
| + | |Reaction Buffer 5x | ||
| + | |50 ul | ||
| + | |- | ||
| + | |Enzyme (5000u/ml) need 1 unit | ||
| + | |1 ul | ||
| + | |- | ||
| + | | Nuclease free water | ||
| + | |159 ul | ||
| + | |} | ||
| + | '''PCR Rxn time Cycle''' | ||
| + | |||
| + | {| border="1" align="center" style="text-align:center;" | ||
| + | |Initial warm up | ||
| + | |94 degrees, 30 secs | ||
| + | |- | ||
| + | |Denaturing | ||
| + | |94 degrees, 30 secs | ||
| + | |- | ||
| + | |Annealing | ||
| + | |55degrees, 1 min | ||
| + | |- | ||
| + | |Extension | ||
| + | |68 degrees, 2min 30sec | ||
| + | |- | ||
| + | |Final extension | ||
| + | |5 min | ||
| + | |- | ||
| + | |Stand by | ||
| + | |4 degrees | ||
| + | |} | ||
| + | |||
These PCR products will need to be run on a gel, and if there are bands, the DNA will need to be purified and then cut with PstI to see if the PstI site is now missing. | These PCR products will need to be run on a gel, and if there are bands, the DNA will need to be purified and then cut with PstI to see if the PstI site is now missing. | ||
| Line 49: | Line 93: | ||
3. We re-transformed the products of mutagenesis reactions 3B and 4A into OneShot TOP10 cells along with a negative control and plated the cells on plates containing Ampicillin. | 3. We re-transformed the products of mutagenesis reactions 3B and 4A into OneShot TOP10 cells along with a negative control and plated the cells on plates containing Ampicillin. | ||
| - | + | ||
| + | We plated 100 ul of the transformed cells. | ||
| + | two different plate each one with the dilution and undiluted cells. | ||
| + | |||
| + | Diluted plates have 10 ul of cells + 90 ul of LB broth | ||
| + | |||
| + | Undiluted has 100 ul of cells. | ||
If there are colonies on the 3B and 4A plates they will need to be screened by colony PCR. | If there are colonies on the 3B and 4A plates they will need to be screened by colony PCR. | ||
| Line 64: | Line 114: | ||
{{LabReportBmore}} | {{LabReportBmore}} | ||
| + | Members Present | ||
| + | Dr. Goode, Dr. Tom, Dr. Lisa, Bivor, Steve, Michelle | ||
| + | |||
| + | Bivor: 8.5 hrs (2-11.30) | ||
Latest revision as of 21:09, 5 August 2011
1. On Sunday, 3/31/11 we had transformed the products of the mutagenesis reaction into bacteria using two protocols: C2987 Transformation (which used store-bought NEB competent cells cat # C2987) or TSS transformation (where we made cells competent using the protocol that Dr. Burkett supplied). We used a positive control (pUC19 plasmid DNA) and a negative control (no DNA).
Results: For the C2987 Transformation protocol there was a lawn of cells for the positive control. There were two plates marked negative control, one had 9 colonies, the other had none. None of the other plates had colonies.
For the TSS Transformation protocol, there was a lawn of cells for the positive control. There was one colony from the transformation of the 3B mutagenesis reaction and one colony from the transformation of the 4A mutagenesis reaction. None of the other plates had colonies.
Based on these results, we decided to do two things:
- screen the two colonies (one 3B and one 4A) that we got from the TSS transformation by colony PCR because they might be what we want
- repeat the transformation using OneShot TOP10 competent cells that were in the -80 freezer to try to get more colonies to screen by colony PCR
2. We set up PCR to screen the two colonies. We set up 4 reactions using different template DNAs:
- Cells from the 4A colony
- Cells from the 3B colony
- No DNA template
- The pET-Taq plasmid that was our template for mutagenesis (positive control)
PCR Recipe
| Template DNA | 1 ul |
| Primer Pol Taq Fwd prefix | 1 uM , 0.3 ul |
| Primer Pol Taq Rvs suffix | 1um , 0.3ul |
| dNTPs | 200uM, 1ul |
| Reaction Buffer 5x | 10ul |
| Enzyme (5000u/ml) need 1 unit | 0.2ul |
| Nuclease free water | 31.8 ul |
| Total Volume of Rxn | 50 ul |
Recipe for Mastermix
| Fwd primer | 15 ul |
| Rv primer | 15 ul |
| dNTPs | 5 ul |
| Reaction Buffer 5x | 50 ul |
| Enzyme (5000u/ml) need 1 unit | 1 ul |
| Nuclease free water | 159 ul |
PCR Rxn time Cycle
| Initial warm up | 94 degrees, 30 secs |
| Denaturing | 94 degrees, 30 secs |
| Annealing | 55degrees, 1 min |
| Extension | 68 degrees, 2min 30sec |
| Final extension | 5 min |
| Stand by | 4 degrees |
These PCR products will need to be run on a gel, and if there are bands, the DNA will need to be purified and then cut with PstI to see if the PstI site is now missing.
3. We re-transformed the products of mutagenesis reactions 3B and 4A into OneShot TOP10 cells along with a negative control and plated the cells on plates containing Ampicillin.
We plated 100 ul of the transformed cells.
two different plate each one with the dilution and undiluted cells.
Diluted plates have 10 ul of cells + 90 ul of LB broth
Undiluted has 100 ul of cells.
If there are colonies on the 3B and 4A plates they will need to be screened by colony PCR.
4. We determined the identity of the promoter, terminator and RBS that were previously transformed into bacteria.
- Promoter is part J23119 in plasmid pSB1A2 (Plate 1 well 18A)
- RBS is part B0030 in plasmid pSB1A2 (Plate 1 well 1H)
- Terminator is part B0015 in plasmid pSB1AK3 (Plate 1 well 23L)
Because none of those parts contained an inducible promoter, we also transformed two more parts from the registry that have an inducible promoter. We resuspended the DNA for these parts in 10 uL water and transformed 2 uL into OneShot TOP10 cells and then plated onto plates containing chloramphenicol.
- Composite part BBa_K314104 (Plate 4 well 5I)
- Composite part BBa_K314103 (Plate 4 well 5K)
Hours
Members Present Dr. Goode, Dr. Tom, Dr. Lisa, Bivor, Steve, Michelle
Bivor: 8.5 hrs (2-11.30)
