Team:Lethbridge/Notebook/Lab Work/August

From 2010.igem.org

(Difference between revisions)
(August 3/2010 Evening)
(August 4/2010)
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  15 (&micro;L) pDNA in plasmid, and 15 (&micro;L) of pDNA biobrick)<br>
  15 (&micro;L) pDNA in plasmid, and 15 (&micro;L) of pDNA biobrick)<br>
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==August 4/2010==
+
==August 4, 2010==
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(in Lab: JV)<br>
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(in lab: JV)<br>
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<b> Objective:</B> PCR analysis of ligation product of aug 3/2010<br>
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<b> Objective:</B> PCR analysis of ligation product of August 3, 2010<br>
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+
* <u>Ligations</u>
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will PCR:<br>
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* <b>ligations</b>
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** pBAD-mRBS
** pBAD-mRBS
** pBAD-SRBS
** pBAD-SRBS
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** dt-pSBIC3
** dt-pSBIC3
** pLacI-SRBS
** pLacI-SRBS
-
 
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* <u>Controls</u>
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* <b>controls</b>
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** pBAD
** pBAD
** TetR
** TetR
** TetR
** TetR
** pLacI
** pLacI
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** Mms6
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** mms6
 +
<b>Method:</b><br>
 +
<u>PCR:</u> Thermocycler set to iGEM program 7<br>
<table border ="3">
<table border ="3">
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<tr><td>           <td><b>1X(&micro;L)</b><td><b>Master Mix(x16)(&micro;L)</b>
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<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x16)(&micro;L)</b>
<tr><td>Milli-Q H<sub>2</sub>O<td>41.8<td>668.6
<tr><td>Milli-Q H<sub>2</sub>O<td>41.8<td>668.6
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>5<td>80
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>5<td>80
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<tr><td>Reverse Primers<td>0.5<td>8
<tr><td>Reverse Primers<td>0.5<td>8
<tr><td>Template DNA<td>1<td>16
<tr><td>Template DNA<td>1<td>16
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<tr><td>PFu polymerase<td>0.2<td>3.2
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<tr><td>Pfu polymerase<td>0.2<td>3.2
</table><br>
</table><br>
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<b>Objective:</b> Run PCR samples on a 2.5% agarose gel(1x TAE)for 70 minutes<br>
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<u>2.5% agarose gel(1x TAE)</u><br>
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<table border ="3">
<table border ="3">
<tr><td>lane<td><b>contents</b><td><b>Successful Ligation ?</b>
<tr><td>lane<td><b>contents</b><td><b>Successful Ligation ?</b>
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<tr><td>14<td>pBad-mRBS<td>x
<tr><td>14<td>pBad-mRBS<td>x
<tr><td>15<td>pBad control<td>---
<tr><td>15<td>pBad control<td>---
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</table><br>
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</table>
 +
*Ran at 100V for 70 minutes.
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all ligations were transformed<br>
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<b>Results:</b> <font color ="red">AGAROSE GEL PICTURE</font><br><br><br>
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<b>Objective:</b> Transform the successful ligations<br>
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<b>Objective:</b> Transform<br>
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<b>Method:</b> used [[Team:Lethbridge/Notebook/Protocols|Competent Cell Transformation]] protocol
<b>Method:</b> used [[Team:Lethbridge/Notebook/Protocols|Competent Cell Transformation]] protocol
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<font color ="red">"AUG 4" is not yet done</font>
<font color ="red">"AUG 4" is not yet done</font>
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==August 4/2010==
==August 4/2010==

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Back to Notebook
Back to Lab Work

Contents

August

August 3, 2010

(in Lab: HB, AV, JV)

Objective: To restrict pBAD, sRBS, mRBS, TetR, dT and pTet for the assembly of pBAD-sRBS-TetR-dT-pTet

Method: Used Restriction of Plasmid DNA protocol.

  • A front verctor was made made in sRBS ,mRBS, dT plasmids using EcoRI and Xbal enzymes
  • pDNA that was cut out of plasmid for a front vector was pBAD, TetR, dT and pLacI using EcoRI and SpeI enzymes
  • A back vector was made in sRBS and mRBS plasmids with PstI and SpeI
  • pDNA that was cut out of plasmid for a back vector was TeTR and it was restricted with XbaI and PstI
ConstructpDNAbufferEnzymes
pBAD-sRBS/mRBSpBADRedEcoRI and SpeI
pBAD-sRBS/mRBSsRBSOrangeXbaI and EcoRI
pBAD-sRBS/mRBSmRBSOrangeXbaI and EcoRII
sRBS/mRBS-TetRsRBSRedPstI and SpeI
sRBS/mRBS-TetRmRBSRedPstI and SpeI
sRBS/mRBS-TetRTetRTangoXbaI and PstI
TetR-dTTetRRedEcoRI and SpeI
TetR-dTdTOrangeXbaI and EcoRI
dT-pTetdTRedEcoRI and SpeI
dT-pTetpTetOrangeXbaI and EcoRI
pLAcI-sRBS/mRBSpLacIRedEcoRI and SpeI
pLAcI-sRBS/mRBSsRBSOrangeXbaI and EcoRI
pLAcI-sRBS/mRBSmRBSOrangeXbaI and EcoRI
Mms6-PET28(a)PET28(a)OrangeNotI
  • For all reactions
    • 158 (µL) Milli-Q H2O
    • 10 (µL) Buffer
    • 0.5(µL) of each enzyme
    • 10 (µL) pDNA

Restriction was incubated for 1 hour at 37oC


Objective: Run PCR of pBAD, TetR, dT, pLacI, and mms6.
Method: Used PCR thermocycler iGEM program 7

ComponentVolume per tube (µL)Master Mix (x6)
MilliQ H2O41.8250.8
10x Pfu Buffer with MgSO4530
dNTPs16
Forward Primer0.53
Reverse Primer0.53
Template DNA1-
Pfu DNA polymerase0.2-
Total50292.8
  • Added 48.8 µL of Master Mix to each PCR reaction


Objective: Complete maxipreps of Lumazine (K249002), EYFP (E0030), and ECFP (E0020) and run on 1% agarose gel.
Method:

LaneSampleComponents (µL)
11 kb ladder2 loading dye (5x) + 0.5 ladder + 3.5 MilliQ H2O
2ECFP2 DNA + 2 loading dye (5x) + 2 MilliQ H2O
3EYFP2 DNA + 2 loading dye (5x) + 2 MilliQ H2O
4Lumazine2 DNA + 2 loading dye (5x) + 2 MilliQ H2O
  • Ran at 100V for 42 minutes. Stained in EtBr for 15 minutes.

Results: AGAROSE GEL PICTURE


Objective: Ligate dT into pSB1C3.
Method:

  • PCR amplify and purify both pSB1C3 and dT
  • Restrict both with EcoRI and PstI
  • Restrict pSB1C3 with DpnI
  • Ligate pSB1C3 and dT

Restriction:

RestrictionMilliQ H2O (µL)Buffer Orange (µL)pDNA (µL)Enzyme (µL)
pSB1C379.2510100.25 EcoRI + 0.25 PstI + 0.25 DpnI
pSB1C3 control801010-
dT79.5010100.25 EcoRI + 0.25 PstI
dT control801010-
  • Restriction were incubated at 37oC for 90 minutes.
  • Enzymes were heat killed for 20 minutes at 80oC.

August 3, 2010 Evening

(in lab: KG, JS)

Objective: To run 1.5% agarose of restrictions: pBAD, sRBS, mRBS, TetR, dT and pTet for the assembly of pBAD-sRBS-TetR-dT-pTet

Method: Used a 1.5% agarose gel with 2 (µL) of loading dye and 10 (µL) of pDNA.

LaneContentsResult
11kb ladder
2pTet (XbaI, EcoRI)good
3pTet (orange buffer)---
4dT (XbaI, EcoR)no good
5dT (orange buffer)---
6mRBS (SpeI, PstI) good
7mRBS (red buffer)---
8sRBS (SpeI, PstI) good
9sRBS (red buffer)---
10mRBS (XbaI, EcoRI) good
11mRBS (orange buffer)---
12sRBS (XbaI, EcoRl) good
13sRBS (orange buffer)---
14pet28(a) good
15100 bp ladder
16PSB1C3 not good
17PSB1C3 restriction digest---

LaneContentsResult
1TetR (EcorI, SpeI) good
2TetR (red buffer)---
3pLacI (EcoRI, SpeI) good
4pLacI (red buffer)---
5Mms6can not tell
6Mms6 control---
7TetR (Xbal, PstI) good
8TetR (tango buffer)---
9pBAD (EcoRI, SpeI)good
10pBAD (red buffer)---
11dT (EcoRI, SpeI)good
12dT (red buffer)---
13pLacI (2)?
14dT control not good
15dT restriction
16100 bp ladder
17dT PCR product good
18Mms6 PCR product good
19pBAD PCR product good
20pLacI PCR product good


Objective: To ligate: pBAD-sRBS/mRBS, sRBS/mRBS-TetR, TetR-dT, dT-pTet, pLacI-sRBS/mRBS, Mms6-ptet28(a), dT-PSB1C3

Method: Ligation of Plasmid DNA

15 (µL) pDNA in plasmid, and 15 (µL) of pDNA biobrick)

August 4, 2010

(in lab: JV)

Objective: PCR analysis of ligation product of August 3, 2010

  • Ligations
    • pBAD-mRBS
    • pBAD-SRBS
    • SRBS-tetR
    • mRBS-TetR
    • dt-pTet
    • mms6-pET-28a
    • dt-pSBIC3
    • pLacI-SRBS
  • Controls
    • pBAD
    • TetR
    • TetR
    • pLacI
    • mms6

Method:
PCR: Thermocycler set to iGEM program 7

Component1X(µL)Master Mix(x16)(µL)
Milli-Q H2O41.8668.6
10x Pfu Buffer with MgSO4580
dNTPs116
Forward Primer0.58
Reverse Primers0.58
Template DNA116
Pfu polymerase0.23.2

2.5% agarose gel(1x TAE)

lanecontentsSuccessful Ligation ?
150bp ladder---
2dt pSBIC3---
3dt pTetx
4dt control---
5sRBS-TetRx
6mRBS-TetR?
7TetR control---
8pLacI-mRBSx
9pLacI-sRBS?
10pLacI control---
11Mms6 pET-28ano band
12Mms6 control---
13pBad-SRBSx
14pBad-mRBSx
15pBad control---
  • Ran at 100V for 70 minutes.

Results: AGAROSE GEL PICTURE


Objective: Transform the successful ligations

Method: used Competent Cell Transformation protocol

  • changes:
    • used 50µL aliquottes of DH5&alpha
    • did not pipette up and down once, the cells were just swirled 3 times
    • added 400µL SOC media, shoock at 370C for 90 min
    • platted 250µL and 150µL

Incubated from 12:00AM to 4;00 PM

results
contents&250µL150µL
dt-pTet:)x
- controlxx
mms6-pET-28a:):)
dt-pSBIC3xx
mRBS-TetR:):)
pLacI-mRBS:):)
SRBS-TetRxx
pBAD-SRBS:):)
+ contol:):)

"AUG 4" is not yet done

August 4/2010

Objective: To run 1.5% agarose of restrictions: pBAD, sRBS, mRBS, TetR, dT and pTet for the assembly of pBAD-sRBS-TetR-dT-pTet

Method: Used a 1.5% agarose gel with 2 (µL) of loading dye and 10 (µL) of pDNA.