Team:University of Sheffield /Wet Lab

From 2008.igem.org

(Difference between revisions)
(→Plasmids and Primers)
(→Plasmids and Primers)
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|-
|-
|BIOBRICK 1 + 2
|BIOBRICK 1 + 2
-
CsrA with promoter,
+
CsrA with promoter
-
and prefix + suffix
+
+prefix
-
(p&s from now on)
+
|Forward:
|Forward:
5' - GAA TTC GCG GCC GCT TCT AGA ACA GAA TGT AAT GCC ATG AC - 3'   
5' - GAA TTC GCG GCC GCT TCT AGA ACA GAA TGT AAT GCC ATG AC - 3'   
Line 112: Line 111:
|-
|-
|CsrA without
|CsrA without
-
promoter + p&s
+
promoter +prefix
|Forward:
|Forward:
5' - GAA TTC GCG GCC GCT TCT AGA TGC TGA TTC TGA CTC GTC GAG TTG - 3'   
5' - GAA TTC GCG GCC GCT TCT AGA TGC TGA TTC TGA CTC GTC GAG TTG - 3'   
Line 130: Line 129:
|BIOBRICK 3 + 4
|BIOBRICK 3 + 4
CrsB with
CrsB with
-
promoter +
+
promoter +prefix
-
p&s
+
|Forward:
|Forward:
5' - GAA TTC GCG GCC GCT TCT AGG TCG ACA GGG AGT CAG ACA AC - 3'   
5' - GAA TTC GCG GCC GCT TCT AGG TCG ACA GGG AGT CAG ACA AC - 3'   
Line 140: Line 138:
|-
|-
|CsrB without
|CsrB without
-
promoter +
+
promoter +prefix
-
p&s
+
|Forward:
|Forward:
5' - GAA TTC GCG GCC GCT TCT AGA GGG AGT CAG ACA ACG AAG T - 3'   
5' - GAA TTC GCG GCC GCT TCT AGA GGG AGT CAG ACA ACG AAG T - 3'   
Line 150: Line 147:
|-
|-
|
|
-
|Reverse for both 3 & 4:
+
|Reverse for both 3 & 4
 +
+suffix:
5' - CTG CAG CGG CCG CTA CTA GTA AAT AAA AAA AGG GAG CAC TG - 3'   
5' - CTG CAG CGG CCG CTA CTA GTA AAT AAA AAA AGG GAG CAC TG - 3'   
Line 156: Line 154:
|GC Content:  48.8%   
|GC Content:  48.8%   
|Molecular Weight (Calculated):  12685.3
|Molecular Weight (Calculated):  12685.3
 +
|-
 +
|BIOBRICKS 5 & 6
 +
UvrY with
 +
promoter +prefix
 +
|Forward:
 +
5' - GAA TTC GCG GCC GCT TCT AGA ATG ACT AAC TAT CAG TAG CGT TAT C - 3' 
 +
|TM (50mM NaCL):  65.5 °C 
 +
|GC Content:  45.7% 
 +
|Molecular Weight (Calculated):  14124.2
 +
|-
 +
|UvrY without
 +
promoter +prefix
 +
|Forward:
 +
5' - GAA TTC GCG GCC GCT TCT AGT ATT CCT TTG ATC AAC GTT CTA C - 3' 
 +
 +
|TM (50mM NaCL):  65.7 °C 
 +
|GC Content:  46.5% 
 +
|Molecular Weight (Calculated):  13110.5
 +
|-
 +
|
 +
|Reverse for both 5 & 6
 +
+suffix
 +
5' - CTG CAG CGG CCG CTA CTA GTA TCA CTG ACT TGA TAA TGT CT - 3' 
 +
 +
|TM (50mM NaCL):  66.3 °C 
 +
|GC Content:  48.8% 
 +
|Molecular Weight (Calculated):  12551.2
|}
|}

Revision as of 00:46, 29 October 2008

UniShefBanner.jpg


Introduction Our project Modelling Wet Lab Our team Timetable Miscellaneous


Introduction Protocols


Wet Lab

Wet lab sessions were where the theory now needed to come into practise. As mainly first years however, there was training to be done before we could get started.

Click here, or on the navigation bar, for the overall timetable of our wet lab sessions.

Click here, or on the sub-navigation bar, for almost all the protocols we used in our wet lab sessions.

The lab books below contain the more detailed jobs carried out by each member of the team.

Lab Books

Dimtry's Lab Book

Eva's Lab Book

Gosia's Lab Book

Hammad's Lab Book

Rosie's Lab Book

Sam's Lab Book


Plasmids and Primers

Experimental Primers
BarA homology

sequences on pKD13 Kanamycin resistant cassette

Forward:

5' - TGA TGA TTC TGA TCC TGG CAC CGA CCG TCC TTA TTG ATT CCG GGG ATC CGT CGA CC - 3'

TM (50mM NaCL): 71.8 °C GC Content: 55.4% Molecular Weight (Calculated): 17149.1
Reverse:

5' - CGT TGA CTT CGG GCG TCA CGA CGC GAG AGG AAA TAC GTG TAG GCT GGA GCT GCT TC - 3'

TM (50mM NaCL): 72.6 °C GC Content: 58.9% Molecular Weight (Calculated): 17377.2
RFP insertion

into PGA operon = PGA homolgy on RFP cassette

Forward:

5' - TAA TTA TAC TCA CCA GCA TCA GGA GAT ATT TAT TTC CAT TAC GTA ACA TAT TTA TCC TTA TTA TTA AGC TAC TAA AGC GT - 3'

TM (50mM NaCL): 65.6 °C GC Content: 28.8% Molecular Weight (Calculated): 24492
Reverse:

5' - AAC TGG CGC GGT TTT GCT GGA TTC GGT TAT GCC GAT GGA CAA TTT AGC GAA GGA AAA GGG ATG GCT TCC TCC GAA GAC GT - 3'

TM (50mM NaCL): 73.3 °C GC Content: 51.2% Molecular Weight (Calculated): 24870.1



Biobrick Primers
BIOBRICK 1 + 2

CsrA with promoter +prefix

Forward:

5' - GAA TTC GCG GCC GCT TCT AGA ACA GAA TGT AAT GCC ATG AC - 3'

TM (50mM NaCL): 66.6 °C GC Content: 48.8% Molecular Weight (Calculated): 12618.2
CsrA without

promoter +prefix

Forward:

5' - GAA TTC GCG GCC GCT TCT AGA TGC TGA TTC TGA CTC GTC GAG TTG - 3'

TM (50mM NaCL): 68.8 °C GC Content: 53.3% Molecular Weight (Calculated): 13850
Reverse for both 1 & 2:

5' - CTG CAG CGG CCG CTA CTA GTA GTA ACT GGA CTG CTG GGA TTT TT - 3'

TM (50mM NaCL): 69 °C GC Content: 52.3% Molecular Weight (Calculated): 13569.8
BIOBRICK 3 + 4

CrsB with promoter +prefix

Forward:

5' - GAA TTC GCG GCC GCT TCT AGG TCG ACA GGG AGT CAG ACA AC - 3'

TM (50mM NaCL): 69.9 °C GC Content: 58.5% Molecular Weight (Calculated): 12660.2
CsrB without

promoter +prefix

Forward:

5' - GAA TTC GCG GCC GCT TCT AGA GGG AGT CAG ACA ACG AAG T - 3'

TM (50mM NaCL): 69 °C GC Content: 55% Molecular Weight (Calculated): 12395.1
Reverse for both 3 & 4

+suffix: 5' - CTG CAG CGG CCG CTA CTA GTA AAT AAA AAA AGG GAG CAC TG - 3'

TM (50mM NaCL): 66.5 °C GC Content: 48.8% Molecular Weight (Calculated): 12685.3
BIOBRICKS 5 & 6

UvrY with promoter +prefix

Forward:

5' - GAA TTC GCG GCC GCT TCT AGA ATG ACT AAC TAT CAG TAG CGT TAT C - 3'

TM (50mM NaCL): 65.5 °C GC Content: 45.7% Molecular Weight (Calculated): 14124.2
UvrY without

promoter +prefix

Forward:

5' - GAA TTC GCG GCC GCT TCT AGT ATT CCT TTG ATC AAC GTT CTA C - 3'

TM (50mM NaCL): 65.7 °C GC Content: 46.5% Molecular Weight (Calculated): 13110.5
Reverse for both 5 & 6

+suffix 5' - CTG CAG CGG CCG CTA CTA GTA TCA CTG ACT TGA TAA TGT CT - 3'

TM (50mM NaCL): 66.3 °C GC Content: 48.8% Molecular Weight (Calculated): 12551.2
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