Team:Lethbridge/Notebook/Lab Work/August

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(Difference between revisions)
(→August)
(→August 3/2010 Evening)
Line 135: Line 135:
<tr><td>3<td>pLacI (EcoRI, SpeI)<td> good
<tr><td>3<td>pLacI (EcoRI, SpeI)<td> good
<tr><td>4<td>pLacI (red buffer)<td>---
<tr><td>4<td>pLacI (red buffer)<td>---
-
<tr><td>5<td>mms6<td>can not tell
+
<tr><td>5<td>Mms6<td>can not tell
-
<tr><td>6<td>mms6 control<td>---
+
<tr><td>6<td>Mms6 control<td>---
<tr><td>7<td>TetR (Xbal, PstI)<td> good
<tr><td>7<td>TetR (Xbal, PstI)<td> good
<tr><td>8<td>TetR (tango buffer)<td>---
<tr><td>8<td>TetR (tango buffer)<td>---
Line 148: Line 148:
<tr><td>16<td>100 bp ladder<td>
<tr><td>16<td>100 bp ladder<td>
<tr><td>17<td>dT PCR product<td> good
<tr><td>17<td>dT PCR product<td> good
-
<tr><td>18<td>mms6 PCR product<td> good
+
<tr><td>18<td>Mms6 PCR product<td> good
<tr><td>19<td>pBAD PCR product<td> good
<tr><td>19<td>pBAD PCR product<td> good
<tr><td>20<td>pLacI PCR product<td> good
<tr><td>20<td>pLacI PCR product<td> good
</table><br>
</table><br>
 +
 +
----
 +
<b>Objective:</b> To ligate: pBAD-sRBS/mRBS, sRBS/mRBS-TetR, TetR-dT, dT-pTet, pLacI-sRBS/mRBS, Mms6-ptet28(a), dT-PSB1C3<br>
 +
 +
<b>Method:</b>ligation protocol
 +
15 (&micro;L) pDNA in plasmid, and 15 (&micro;L) of pDNA biobrick)<br>
 +
==August 4/2010==
==August 4/2010==
(in Lab: JV)<br>
(in Lab: JV)<br>

Revision as of 02:38, 27 August 2010

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Back to Notebook
Back to Lab Work

Contents

August

August 3/2010

(In Lab: HB)

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 370C


Harland to read over and make sure what i wrote is correct

August 3/2010 Evening

(In Lab:K.G J.S)

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 protocol 

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

August 4/2010

(in Lab: JV)

Objective: PCR analysis of ligation product of aug 3/2010

will PCR:

  • ligations
    • pBAD-mRBS
    • pBAD-SRBS
    • SRBS-tetR
    • mRBS-TetR
    • dt-pTet
    • mms6-pET-28a
    • dt-pSBIC3
    • pLacI-SRBS
  • controls
    • pBAD
    • TetR
    • TetR
    • pLacI
    • Mms6
1X(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

Objective: Run PCR samples on a 2.5% agarose gel(1x TAE)for 70 minutes

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---

all ligations were transformed

Objective: Transform

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.

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