Team:Exeter/lab book/gibs/wk4
From 2012.igem.org
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<p><b><u>Operon Construction: 30th July - 3rd August 2012</u></b></p> | <p><b><u>Operon Construction: 30th July - 3rd August 2012</u></b></p> | ||
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<!<p><b>**Monday 30.7.12**</b></p><br> | <!<p><b>**Monday 30.7.12**</b></p><br> | ||
<p><b>4 pm</b></p> | <p><b>4 pm</b></p> | ||
- | <p>Inoculated LB(kan) broth with transformed pBAD large (BBa_I0500) colonies <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/2" style="color:# | + | <p>Inoculated LB(kan) broth with transformed pBAD large (BBa_I0500) colonies <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/2" style="color:#57b947"><u>according to protocol</u></a></p><br> |
<p><b>**Tuesday 31.7.12**</b></p> | <p><b>**Tuesday 31.7.12**</b></p> | ||
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<p><b><i>Step 10</i></b>20µl milli-Q water added in place of elution buffer, followed by another 10 µl after incubation and centrifugation</p><br> | <p><b><i>Step 10</i></b>20µl milli-Q water added in place of elution buffer, followed by another 10 µl after incubation and centrifugation</p><br> | ||
- | <p><a href="https://2012.igem.org/Team:Exeter/lab_book/proto/4" style="color:# | + | <p><a href="https://2012.igem.org/Team:Exeter/lab_book/proto/4" style="color:#57b947"><u>3A BioBrick Assembly </u></a> for pBAD-RBS (BBa_I0500 + BBa_B0034)</p><br> |
<p><u>Digestion:</u></p><br> | <p><u>Digestion:</u></p><br> | ||
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- | <p>Destination plasmid (pSB1K3 – kanamycin resistance) digested according to <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/10" style="color:# | + | <p>Destination plasmid (pSB1K3 – kanamycin resistance) digested according to <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/10" style="color:#57b947"><u> protocol for linearised backbone</u></a></p> |
<p>Digested at 37 °C for 1 hour</p> | <p>Digested at 37 °C for 1 hour</p> | ||
<p>Heat inactivated at 80 °C for 20 minutes</p><br> | <p>Heat inactivated at 80 °C for 20 minutes</p><br> | ||
- | <p> Ligation according to same <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/10" style="color:# | + | <p> Ligation according to same <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/10" style="color:#57b947"><u>protocol</u></a></p> |
<p>Downstream digest: 2 µl</p> | <p>Downstream digest: 2 µl</p> | ||
<p>Plasmid digest: 2 µl</p> | <p>Plasmid digest: 2 µl</p> | ||
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<p><b>3 pm</b></p> | <p><b>3 pm</b></p> | ||
- | <p>pBAD-RBS construct <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/1" style="color:# | + | <p>pBAD-RBS construct <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/1" style="color:#57b947"><u>transformed</u></a></p> |
<p>2 µl of construct DNA added to competent cells | <p>2 µl of construct DNA added to competent cells | ||
<p>Plated volumes were 50 µl and 150 µl on LB(kan) </p><br> | <p>Plated volumes were 50 µl and 150 µl on LB(kan) </p><br> |
Revision as of 10:29, 26 September 2012
Operon Construction: 30th July - 3rd August 2012 **Monday 30.7.12**4 pm Inoculated LB(kan) broth with transformed pBAD large (BBa_I0500) colonies according to protocol **Tuesday 31.7.12** 9 am Centrifuged broth tubes for 5 minutes, 3901 rcf, 4 °C Supernatant discarded Miniprepof pBAD (BBa_I0500) Step 4 changed to 9 minutes
Step 1020µl milli-Q water added in place of elution buffer, followed by another 10 µl after incubation and centrifugation 3A BioBrick Assembly for pBAD-RBS (BBa_I0500 + BBa_B0034) Digestion: Upstream pBAD (BBa_I0500) plasmid DNA: 8.68 µl MilliQ water: 18 µl Downstream RBS (BBa_B0034) plasmid DNA: 2.94 µl MilliQ water: 14.56 Destination plasmid (pSB1K3 – kanamycin resistance) digested according to protocol for linearised backbone Digested at 37 °C for 1 hour Heat inactivated at 80 °C for 20 minutes Ligation according to same protocol Downstream digest: 2 µl Plasmid digest: 2 µl Water: 2.5 µl Incubated at room temperature for 1.5 hours Heat inactivated at 80 °C for 20 minutes 2pm Sent DNA off for sequencing with VF2 and VR primers – pBAD (BBa_I0500) according to Bioline instructions 3 pm pBAD-RBS construct transformed 2 µl of construct DNA added to competent cells Plated volumes were 50 µl and 150 µl on LB(kan) **Thursday 2.8.12** 9 am Broth tubes centrifuged for 5 minutes, 3901 rcf, 4 °C Supernatant discarded Miniprepof pBAD-RBS (BBa_I0500 + BBa_B0034) Step 1045µl milli-Q water added in place of elution buffer, followed by another 5 µl after incubation and centrifugation 2.30 pm Gel of pPAD large-RBS (BBa_I0500 + BBa_B0034) Digest for gel (using only one enzyme – nanodrop data was uncertain) DNA (assumed ~100ng/µl): 5 µl EcoRI-HF: 1µl 100X BSA: 0.5 µl 10X NEBuffer 2: 5 µl Water: 11 µl Incubated at 37 °C for 20 minutes Run on 1% agarose gel LB(amp) streak plates made for pBAD-RBS (BBa_I0500 + BBa_B0034) dregs from miniprep. |