October
October 1, 2010
KG
Objective: PCR of xylE for conformation of transformation.
| Component | 1X(µL) | Master Mix(x24)(µL)
|
| Milli-Q H2O | 13.8 | 331.2
|
| 10x Pfu Buffer with MgSO4 | 2 | 48
|
| dNTPs | 1 | 24
|
| Forward VF2 Primer | 1 | 24
|
| Reverse VR Primer | 1 | 24
|
| Template DNA | 1 |
|
| Pfu polymerase | 0.2 | 4.8
|
Ran with PFV program on thermocycler with extension time of 3 minutes.
October 2, 2010
ADS
Objective: Purify plasmid DNA from transformed Mr. GENE synthesized DNA
- <partinfo>K331022</partinfo>
- <partinfo>K331023</partinfo>
- <partinfo>K331024</partinfo>
- <partinfo>K331025</partinfo>
Method: Use Qiagen miniprep method with BioBasic EZ-10 Spin columns.
Objective: Create 20 new biobricks using recently arrived synthesized DNA and terminator <partinfo>B0015</partinfo>, pLac <partinfo>R0010</partinfo>, and pTet <partinfo>R0040</partinfo> to create the following new biobricks:
- 1)R0010 + K331022 to obtain <partinfo>K331026</partinfo>
- 2)R0040 + K331022 to obtain <partinfo>K331030</partinfo>
- 3)K331022 + B0015 to obtain <partinfo>K331034</partinfo>
- 4)K331022 + K331024 to obtain <partinfo>K331038</partinfo>
- 5)R0010 + K331023 to obtain <partinfo>K331027</partinfo>
- 6)R0040 + K331023 to obtain <partinfo>K331031</partinfo>
- 7)K331023 + B0015 to obtain <partinfo>K331035</partinfo>
- 8)K331023 + K331025 to obtain <partinfo>K331039</partinfo>
- 9)R0010 + K331024 to obtain <partinfo>K331028</partinfo>
- 10)R0040 + K331024 to obtain <partinfo>K331032</partinfo>
- 11)K331024 + B0015 to obtain <partinfo>K331036</partinfo>
- 12)K331024 + K331022 to obtain <partinfo>K331040</partinfo>
- 13)R0010 + K331025 to obtain <partinfo>K331029</partinfo>
- 14)R0040 + K331025 to obtain <partinfo>K331033</partinfo>
- 15)K331025 + B0015 to obtain <partinfo>K331037</partinfo>
- 16)K331025 + K331023 to obtain <partinfo>K331041</partinfO>
- 17)K331022 to obtain <partinfo>K331022</partinfo>
- 18)K331023 to obtain <partinfo>K331023</partinfo>
- 19)K331024 to obtain <partinfo>K331024</partinfo>
- 20)K331025 to obtain <partinfo>K331025</partinfo>
All parts will be inserted into pSB1C3.
Method: Use BioBrick Assembly Method
Results: Obtained positive colonies from all transformations.
HB
Objective: PCR mms6 from Mr. Gene to attach fusion standard to the N-term.
| Component | 1X(µL) | Master Mix(x6.5)(µL)
|
| Milli-Q H2O | 12.8 | 83.2
|
| 10x Pfu Buffer with MgSO4 | 2 | 13
|
| dNTPs | 1 | 6.5
|
| mms6 Prefix Fusion Forward Primer | 1 | 6.5
|
| Standard Suffix Reverse Primer | 1 | 6.5
|
| Template DNA | 2 | 13
|
| Pfu polymerase | 0.2 |
|
Prefix fusion sense primer has melting temperature of 56.1oC. Standard suffix primer has melting temperature of 61.3oC.
PCR- Conditions:
1. 95oC for 180 sec
2. 95oC for 30 sec
3. 56.3oC for 30 sec
4. 72oC for 150 sec
5. 72oC for 600 sec
(35 cycles)
Gradient -
1. 54.3oC 2. 55.4oC 3. 56.0oC 4. 56.6oC 5. 57.8oC 6. 58.7oC
Objective: PCR mms6 from Mr. Gene to attach fusion standard to the C-term.
| Component | 1X(µL) | Master Mix(x6.5)(µL)
|
| Milli-Q H2O | 12.8 | 83.2
|
| 10x Pfu Buffer with MgSO4 | 2 | 13
|
| dNTPs | 1 | 6.5
|
| Prefix Forward Primer | 1 | 6.5
|
| mms6 Fusion Suffix Reverse Primer | 1 | 6.5
|
| Template DNA | 2 | 13
|
| Pfu polymerase | 0.2 |
|
Prefix fusion sense primer has melting temperature of 58.8oC. Standard suffix primer has melting temperature of 75oC.
PCR- Conditions:
1. 95oC for 180 sec
2. 95oC for 30 sec
3. 53.8oC for 30 sec
4. 72oC for 150 sec
5. 72oC for 600 sec
(35 cycles)
Gradient -
1. 51.4oC 2. 52.3oC 3. 53.5oC 4. 54.1oC 5. 55.3oC 6. 56.2oC
JV
Objective: Analyzed KG and HB's PCRs.
Objective: Run samples on 2% agarose gel at 100V for 70 minutes.
October 5, 2010
ADS
Objective: Purify plasmid DNA from successfully assembled biobricks.
Method: Use Qiagen miniprep method with BioBasic EZ-10 columns.
Will send these minipreps for sequencing at Macrogen.
October 16, 2010
JV
Objective: Separate different fractions of the cytosol of cells containing catechol 2,3-dioxygenase
Method:
- Grow DH5α cells in 500mL of LB media with Amp+.
- Optical density of cell suspension (measured at 600nm): 4.55
- Spun cells down: 10 minutes, 4oC, at 5000RPM
- Cell pellet weight is 3.03g
- Flash froze cells and stored at -80oC
Plasmid DNA was also isolated using QIAGEN spin column protocol. DNA was eluted to 60µL.
October 19, 2010
Js
Objective Ligate biobrick parts
Method restrict and ligate into RFP plasmid using the biobrick standard method then transform cells
- pLacI+mRBS
- mRBS+Lum
- Lum+dT
- K118021+dT
- pBad+mRBS
- mRBS+TetR
- TetR+dT
- forgot to add RFP plasmid at ligation step
- repeated all protocol
- ligated the RFP plasmid into the ligated linear DNA as well
October 21, 2010
Js,Jv
Objective Colony PCR for conformation of ligation
Method pick white colonies and perform colony pcr
- pick white colonies from plates
| Component | 1X(µL) | Master Mix(x24)(µL)
|
| Milli-Q H2O | 6.8 | 261.8
|
| 10x Pfu Buffer with MgSO4 | 2 | 77
|
| dNTPs | 2 | 77
|
| Forward VF2 Primer | 2 | 77
|
| Reverse VR Primer | 2 | 77
|
| Template DNA | 5 |
|
| Pfu polymerase | 0.2 | 77
|
PCR- Conditions:
1. 95oC for 180 sec
2. 95oC for 30 sec
3. 53.8oC for 30 sec
4. 72oC for 150 sec
5. 72oC for 600 sec
(35 cycles)
ran a 2% agarose gel for conformation
- no XYLE(K118021)-dT bands were observed, grew cells overnight.
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