Team:Lethbridge/Notebook/Lab Work/October

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Latest revision as of 23:51, 27 October 2010

Feel free to look around our notebook!

Here you can check out the work we have done in the lab, click on a month to take a look!

Contents

1 October

1.1 October 1, 2010

1.1.1 KG

1.2 October 2, 2010

1.2.1 ADS

1.2.2 HB

1.2.3 JV

1.3 October 5, 2010

1.3.1 ADS

1.4 October 16, 2010

1.4.1 JV

1.5 October 19, 2010

1.5.1 Js

1.6 October 21, 2010

1.6.1 Js,Jv

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 56.1^oC. Standard suffix primer has melting temperature of 61.3^oC.
PCR- Conditions: 1. 95^oC for 180 sec 2. 95^oC for 30 sec 3. 56.3^oC for 30 sec 4. 72^oC for 150 sec 5. 72^oC for 600 sec (35 cycles)
Gradient - 1. 54.3^oC 2. 55.4^oC 3. 56.0^oC 4. 56.6^oC 5. 57.8^oC 6. 58.7^oC

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 58.8^oC. Standard suffix primer has melting temperature of 75^oC.
PCR- Conditions: 1. 95^oC for 180 sec 2. 95^oC for 30 sec 3. 53.8^oC for 30 sec 4. 72^oC for 150 sec 5. 72^oC for 600 sec (35 cycles)
Gradient - 1. 51.4^oC 2. 52.3^oC 3. 53.5^oC 4. 54.1^oC 5. 55.3^oC 6. 56.2^oC

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, 4^oC, at 5000RPM
Cell pellet weight is 3.03g
Flash froze cells and stored at -80^oC

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. 95^oC for 180 sec 2. 95^oC for 30 sec 3. 53.8^oC for 30 sec 4. 72^oC for 150 sec 5. 72^oC for 600 sec (35 cycles)

ran a 2% agarose gel for conformation

no XYLE(K118021)-dT bands were observed, grew cells overnight.

Retrieved from "http://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/October"

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 =<font color="white">October=
+==<font color="white">October 1, 2010==
+===<font color="white">KG===
+<b>Objective:</b> PCR of xylE for conformation of transformation.
+<table border ="3">
+<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x24)(&micro;L)</b>
+<tr><td>Milli-Q H<sub>2</sub>O<td>13.8<td>331.2
+<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>48
+<tr><td>dNTPs<td>1<td>24
+<tr><td>Forward VF2 Primer<td>1<td>24
+<tr><td>Reverse VR Primer<td>1<td>24
+<tr><td>Template DNA<td>1<td>
+<tr><td>Pfu polymerase<td>0.2<td>4.8
+</table><br>
+Ran with PFV program on thermocycler with extension time of 3 minutes.
+==<font color="white">October 2, 2010==
+===<font color="white">ADS===
+<b>Objective:</b> Purify plasmid DNA from transformed Mr. GENE synthesized DNA
+*<partinfo>K331022</partinfo>
+*<partinfo>K331023</partinfo>
+*<partinfo>K331024</partinfo>
+*<partinfo>K331025</partinfo>
+<b>Method:</b> Use Qiagen miniprep method with BioBasic EZ-10 Spin columns.<br>
+---------------------------------------------------------------------------------------------------------------------
+<b>Objective:</b> 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.<br>
+<b>Method:</b> Use BioBrick Assembly Method <br>
+<b>Results:</b> Obtained positive colonies from all transformations.
+----
+===<font color="white">HB===
+<b>Objective:</b> PCR mms6 from Mr. Gene to attach fusion standard to the N-term.<br>
+<table border ="3">
+<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x6.5)(&micro;L)</b>
+<tr><td>Milli-Q H<sub>2</sub>O<td>12.8<td>83.2
+<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>13
+<tr><td>dNTPs<td>1<td>6.5
+<tr><td>mms6 Prefix Fusion Forward Primer<td>1<td>6.5
+<tr><td>Standard Suffix Reverse Primer<td>1<td>6.5
+<tr><td>Template DNA<td>2<td>13
+<tr><td>Pfu polymerase<td>0.2<td>
+</table><br>
+Prefix fusion sense primer has melting temperature of 56.1<sup>o</sup>C.  Standard suffix primer has melting temperature of 61.3<sup>o</sup>C.
+PCR- Conditions:
+.  95<sup>o</sup>C for 180 sec
+.  95<sup>o</sup>C for 30 sec
+.  56.3<sup>o</sup>C for 30 sec
+.  72<sup>o</sup>C for 150 sec
+.  72<sup>o</sup>C for 600 sec
+(35 cycles)
+Gradient -
+.  54.3<sup>o</sup>C  2.  55.4<sup>o</sup>C  3.  56.0<sup>o</sup>C  4.  56.6<sup>o</sup>C  5.  57.8<sup>o</sup>C   6. 58.7<sup>o</sup>C
+----
+<b>Objective:</b> PCR mms6 from Mr. Gene to attach fusion standard to the C-term.<br>
+<table border ="3">
+<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x6.5)(&micro;L)</b>
+<tr><td>Milli-Q H<sub>2</sub>O<td>12.8<td>83.2
+<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>13
+<tr><td>dNTPs<td>1<td>6.5
+<tr><td>Prefix Forward Primer<td>1<td>6.5
+<tr><td>mms6 Fusion Suffix Reverse Primer<td>1<td>6.5
+<tr><td>Template DNA<td>2<td>13
+<tr><td>Pfu polymerase<td>0.2<td>
+</table><br>
+Prefix fusion sense primer has melting temperature of 58.8<sup>o</sup>C.  Standard suffix primer has melting temperature of 75<sup>o</sup>C.
+PCR- Conditions:
+.  95<sup>o</sup>C for 180 sec
+.  95<sup>o</sup>C for 30 sec
+.  53.8<sup>o</sup>C for 30 sec
+.  72<sup>o</sup>C for 150 sec
+.  72<sup>o</sup>C for 600 sec
+(35 cycles)
+Gradient -
+.  51.4<sup>o</sup>C  2.  52.3<sup>o</sup>C  3.  53.5<sup>o</sup>C  4.  54.1<sup>o</sup>C  5.  55.3<sup>o</sup>C   6. 56.2<sup>o</sup>C
+----
+===<font color="white">JV===
+<b>Objective:</b> Analyzed KG and HB's PCRs.<br>
+<b>Objective:</b> Run samples on 2% agarose gel at 100V for 70 minutes.<br>
+==<font color="white">October 5, 2010==
+===<font color="white">ADS===
+<b>Objective:</b> Purify plasmid DNA from successfully assembled biobricks.<br>
+<b>Method:</b> Use Qiagen miniprep method with BioBasic EZ-10 columns.<br>
+Will send these minipreps for sequencing at Macrogen.
+<br>
+==<font color="white">October 16, 2010==
+===<font color="white">JV===
+<b>Objective:</b> Separate different fractions of the cytosol of cells containing catechol 2,3-dioxygenase <br>
+<b>Method:</b>
+*Grow DH5&alpha; cells in 500mL of LB media with Amp<sup>+</sup>.
+*Optical density of cell suspension (measured at 600nm): 4.55
+*Spun cells down: 10 minutes, 4<sup>o</sup>C, at 5000RPM
+*Cell pellet weight is 3.03g
+*Flash froze cells and stored at -80<sup>o</sup>C <br>
+Plasmid DNA was also isolated using QIAGEN spin column protocol. DNA was eluted to 60&micro;L.<br>
+==<font color="white">October 19, 2010==
+===<font color="white">Js===
+<b>Objective</b> Ligate biobrick parts<br>
+<b>Method</b> 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
+==<font color="white">October 21, 2010==
+===<font color="white">Js,Jv===
+<b>Objective</b> Colony PCR for conformation of ligation
+<b>Method</b> pick white colonies and perform colony pcr
+*pick white colonies from plates
+<table border ="3">
+<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x24)(&micro;L)</b>
+<tr><td>Milli-Q H<sub>2</sub>O<td>6.8<td>261.8
+<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>77
+<tr><td>dNTPs<td>2<td>77
+<tr><td>Forward VF2 Primer<td>2<td>77
+<tr><td>Reverse VR Primer<td>2<td>77
+<tr><td>Template DNA<td>5<td>
+<tr><td>Pfu polymerase<td>0.2<td>77
+</table><br>
+PCR- Conditions:
+.  95<sup>o</sup>C for 180 sec
+.  95<sup>o</sup>C for 30 sec
+.  53.8<sup>o</sup>C for 30 sec
+.  72<sup>o</sup>C for 150 sec
+.  72<sup>o</sup>C for 600 sec
+(35 cycles)
+<br>
+<br>
+ran a 2% agarose gel for conformation
+*no XYLE(K118021)-dT bands were observed, grew cells overnight.
 <br>

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

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