IGEM:MIT/2007/Notebook/2007-7-17: Difference between revisions
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==Digestion of CPX for 3A== | ==Digestion of CPX for 3A== | ||
<pre> | <pre> | ||
5 µl CPX (diluted to 1µg/5µl) | |||
0. | 0.5 µl BSA | ||
5 µl NEB3 Buffer | |||
1 µl XbaI | |||
1 µl PstI | |||
37. | 37.5 µl H2O | ||
------------------ | ------------------ | ||
50 µl Total | |||
</pre> | </pre> | ||
==Digestion of (F2620 and B0034) for 3A== | ==Digestion of (F2620 and B0034) for 3A== | ||
<pre> | <pre> | ||
37 µl (F2620 and B0034) @ 27 ng/µl | |||
0. | 0.5 µl BSA | ||
5 µl NEB2 Buffer | |||
1 µl EcoRI | |||
1 µl SpeI | |||
5. | 5.5 µl H2O | ||
------------------ | ------------------ | ||
50 µl Total | |||
</pre> | </pre> | ||
*Digested pSB1AC3: | *Digested pSB1AC3: 15 µl @ 125 ng/µl | ||
*Digestions placed in 37C @ 11am | *Digestions placed in 37C @ 11am | ||
| Line 37: | Line 37: | ||
**In step 2, Note -- TK said that less than 5% of the solution should be cell volume | **In step 2, Note -- TK said that less than 5% of the solution should be cell volume | ||
**Step 4 -- change to 45 to 50 seconds at 42 degrees | **Step 4 -- change to 45 to 50 seconds at 42 degrees | ||
**Step 7 -- The 45 incubation time is okay for Kan and Amp resistances, but should be increased to 1.5 to 2 hours for Tet or | **Step 7 -- The 45 incubation time is okay for Kan and Amp resistances, but should be increased to 1.5 to 2 hours for Tet or Cm resistant cells | ||
===Miniprep Results for 5mL LCs of pT040 made on 7/16=== | ===Miniprep Results for 5mL LCs of pT040 made on 7/16=== | ||
| Line 66: | Line 66: | ||
Ligation Mix: | Ligation Mix: | ||
<pre> | <pre> | ||
1 µl 53ng backbone (pSB1AC3) | |||
6 µl 72ng F+B | |||
6. | 6.2 µl 47ng CPX | ||
0. | 0.5 µl T4 ligase | ||
2. | 2.0 µl 10x T4 ligase buffer | ||
4. | 4.3 µl H20 | ||
-------------------------------- | -------------------------------- | ||
20 µl Total | |||
</pre> | </pre> | ||
Negative Control(Backbone only, no inserts): | Negative Control(Backbone only, no inserts): | ||
<pre> | <pre> | ||
1 µl 53ng backbone (pSB1AC3) | |||
0. | 0.5 µl T4 ligase | ||
2. | 2.0 µl 10x T4 ligase buffer | ||
16. | 16.5 µl H20 | ||
-------------------------------- | -------------------------------- | ||
20 µl Total | |||
</pre> | </pre> | ||
| Line 97: | Line 97: | ||
==FhuA Ligated Insert Transformation== | ==FhuA Ligated Insert Transformation== | ||
* | * 10 ul each of PHIE6 and PICCS8 into TK cells | ||
=AHL RFP Testing= | =AHL RFP Testing= | ||
Failed | Failed | ||
Possible Reasons: | Possible Reasons: | ||
=Mer Construct Digestion= | |||
*2 ul EcoR1 Buffer | |||
*2 10X BSA | |||
*.5 EcoR! | |||
*.5 Spe1 | |||
*9.6 ul DNA | |||
*5.4 H20 | |||
Latest revision as of 11:44, 23 July 2007
Agenda
- Check sequencing results of second (uncontaminated) set of miniprepped 3K3+F2620+B0034
- Digest CPX, 3K3+F2620+B0034 (if sequencing is correct), and a hi-copy plasmid for 3A assembly
- Ligate digested CPX, 3K3+F2620+B0034, and hi-copy plasmid
- Test Standard Assembly of pSB3k3 with F2620, B0034, E1010 using AHL
- LC grown overnight
Digestion of CPX for 3A
5 µl CPX (diluted to 1µg/5µl) 0.5 µl BSA 5 µl NEB3 Buffer 1 µl XbaI 1 µl PstI 37.5 µl H2O ------------------ 50 µl Total
Digestion of (F2620 and B0034) for 3A
37 µl (F2620 and B0034) @ 27 ng/µl 0.5 µl BSA 5 µl NEB2 Buffer 1 µl EcoRI 1 µl SpeI 5.5 µl H2O ------------------ 50 µl Total
- Digested pSB1AC3: 15 µl @ 125 ng/µl
- Digestions placed in 37C @ 11am
Changes to the OWW "Transforming Chemically Competent Cells" Protocol for TK Cells
- All steps are the same, except for the following:
- In step 2, Note -- TK said that less than 5% of the solution should be cell volume
- Step 4 -- change to 45 to 50 seconds at 42 degrees
- Step 7 -- The 45 incubation time is okay for Kan and Amp resistances, but should be increased to 1.5 to 2 hours for Tet or Cm resistant cells
Miniprep Results for 5mL LCs of pT040 made on 7/16
Colony DNA Conc. 1 60.6 ng/µL 2 64.3 ng/µL 3 78.2 ng/µL 4 57.9 ng/µL
eppendorfs containing the purified plasmids are labeled:
iGEM 7/17
pur. pT040
LC #
Concentration
Miniprep of PCR Purified Double Digested CPX and F2620+B0034
CPX (X, P): 7.6 ng/µl F2620+B0034 (E, S): 12.0 ng/µl
Ligation of F2620+B0034 and CPX into pSB1AC3
Ligation Mix:
1 µl 53ng backbone (pSB1AC3) 6 µl 72ng F+B 6.2 µl 47ng CPX 0.5 µl T4 ligase 2.0 µl 10x T4 ligase buffer 4.3 µl H20 -------------------------------- 20 µl Total
Negative Control(Backbone only, no inserts):
1 µl 53ng backbone (pSB1AC3) 0.5 µl T4 ligase 2.0 µl 10x T4 ligase buffer 16.5 µl H20 -------------------------------- 20 µl Total
Put into 16C at 4PM into Thermal Gradient Cycler
Transformations
CPX Transformation
- Transformed 1 ul into TK cells
GFP Tranformation
- Transformed 1 ul into TK cells
FhuA Ligated Insert Transformation
- 10 ul each of PHIE6 and PICCS8 into TK cells
AHL RFP Testing
Failed Possible Reasons:
Mer Construct Digestion
- 2 ul EcoR1 Buffer
- 2 10X BSA
- .5 EcoR!
- .5 Spe1
- 9.6 ul DNA
- 5.4 H20
