IGEM:Imperial/2010/Vectors team: Difference between revisions
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==Schedule & Lab notes== | ==Schedule & Lab notes== | ||
===<font color = #6B3FA0>'''PyrD Vector'''</font>=== | ===<font color = #6B3FA0>'''PyrD Vector'''</font>=== | ||
====Week 6==== | |||
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|- | |||
! Week 6 !! Monday !! Tuesday!! Wednesday !! Thursday !! Friday | |||
|- | |||
| MORNING || || || || || | |||
*<font color = #6B3FA0>Restriction digestion of 5' integration site for the PyrD vector ( Using Spe and Pst) | |||
</font> | |||
|- | |||
| AFTERNOON || || || || | |||
*<font color = #6B3FA0>'''Starting assembly of PyrD vector'''</font> | |||
*<font color = #6B3FA0>Annealing the synthesized oligos ( Pme and diff sites)</font> | |||
|| | |||
*<font color = #6B3FA0>Gel analysis of resultant products from digestion mixture</font> | |||
*<font color = #6B3FA0>PCR purification of digestion mixture</font> | |||
*<font color = #6B3FA0>Further gel analysis</font> | |||
|} | |||
====Week 7==== | |||
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|- | |||
! Week 6 !! Monday !! Tuesday!! Wednesday !! Thursday !! Friday | |||
|- | |||
| MORNING | |||
||<font color = #CA2C92>'''Starting assembly of AmyE vector'''</font> | |||
*<font color = #CA2C92>Restriction digestion of BB k14070 for the AmyE vector (using Eco and Spe)</font> | |||
*<font color = #CA2C92>Restriction digestion of BB k14064 for the AmyE vector (using Eco and Xba)</font> | |||
*<font color = #6B3FA0>Restriction digestion of 5' integration site (k08) for the PyrD vector (using Eco and Spe)</font> | |||
*<font color = #6B3FA0>PCR amplification of vector backbone PSB1C3 for the PyrD vector</font> | |||
|| | |||
*<font color = #6B3FA0>Gel analysis and extraction of 5' int site for PyrD vector (repetition of step due to absence of DNA during gel analysis yesterday)</font> | |||
*<font color = #CA2C92>Gel purification of k70</font> | |||
*<font color = #6B3FA0>Gel purification of k08</font> | |||
*<font color = #CA2C92>Re-analysis of k70, k64 (for AmyE) on gel to work out ratios for ligation set up</font> | |||
*<font color = #6B3FA0>Re-analysis of k08, Pme oligos and pSB1C3 (for PyrD) on gel to work out ratios for ligation set up</font> | |||
*<font color = #6B3FA0>Restriction digestion of pveg promoter (k53) (using Eco and Spe) and spec cassette (k65) (using Xba and Pst) for PyrD vector</font> | |||
*<font color = #6B3FA0>Restriction digestion of pSB1C3 for PyrD (using Eco and Pst)</font> | |||
|| | |||
*<font color = #6B3FA0>Check for transformed colonies (colonies that have taken up the vector with the 5'diff) and prepare for colony PCR</font> | |||
*<font color = #6B3FA0>Gel purification of k53 and k65 for AmyE in preparation for ligations this afternoon</font> | |||
*<font color = #6B3FA0>Gel extraction and re-analysis on gel of k53, k65 and psB1C3 for Spec casette in preparation for ligations this afternoon</font> | |||
|| | |||
*<font color = #CA2C92>Transformation of overnight ligations of: | |||
k64 and k70, k70 only, </font> | |||
<font color = #6B3FA0> | |||
Spec </font> | |||
<font color = #6B3FA0> | |||
and 5' PyrD diff</font> | |||
|| | |||
*<font color = #6B3FA0>Replica plating and colony PCR of: | |||
Spec and 5' PyrD diff</font> | |||
*<font color = #CA2C92>Plate wash of: | |||
k64 and k70. k70 only was discarded since this was purely for a baground check</font> | |||
|- | |||
| AFTERNOON | |||
|| | |||
*<font color = #6B3FA0>PCR purification of PSB1C3 vector </font> | |||
*<font color = #CA2C92>PCR purification of BB k14064 digestion products</font> | |||
*<font color = #CA2C92>Gel analysis of digestion products of BB k14070</font> | |||
*<font color = #CA2C92>Gel extraction of digestion products of BB k14070</font> | |||
|| | |||
*<font color = #CA2C92>Restriction digestion of k64 and subsequent PCR purification (repetition of step due to absence of DNA during gel analysis)</font> | |||
*<font color = #CA2C92>Gel analysis of k70, k64 (for AmyE) to work out ligation ratios</font> | |||
*<font color = #6B3FA0>Gel analysis and extraction of k53 and k65</font> | |||
*<font color = #6B3FA0>Bench (1 hour) and overnight ligation of 5'diff with the pSB1C3 vector</font> | |||
*<font color = #6B3FA0>Transformation of E.Coli with the bench ligated vector</font> | |||
|| | |||
*<font color = #CA2C92>Dephosphorylation of k64 and set up of overnight ligations for k64 and k70 (vector and insert) and k64 (vector)only (for negative control; check of background)</font> | |||
*<font color = #6B3FA0>Set up overnight ligations of SpecR</font> | |||
|| | |||
|| | |||
*<font color = #6B3FA0>Gel analysis of colony PCR products of Spec and 5' PyrD diff</font> | |||
* Annealing of diff P oligos (used for both PyrD and AmyE vectors) | |||
|} | |||
===Week 6=== | ===Week 6=== | ||
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Revision as of 08:17, 5 September 2010
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<meta http-equiv="Content-Type" content="text/html; charset=utf-8" /> <title>Imperial iGEM 2010</title> <script type="text/javascript" src="http://ajax.googleapis.com/ajax/libs/jquery/1.4.2/jquery.min.js"></script> <script type="text/javascript"> $(document).ready(function(){ $("ul[id|=poplist]").hide();
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<p id="popshow">Follow our progress: Click me!</p> <div id="popcon"> <ul id="poplist"> <li><a href="#Objectives" class="popt">Objectives</a></li> <li><a href="#Assembly_of_Vectors" class="popt">Assembly of Vectors</a></li> <li><a href="#Schedule" class="popt">Schedule</a></li> <li><a href="#Lab_Notes" class="popt">Lab Notes</a></li>
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Objectives
We are assembling the AmyE and PyrD vectors in order to transform B. Subtilis with our parts. Once completed, these vectors will be reusable and can then be used to introduce any relevant piece of DNA directly into B.subtilis genome. The vectors will be used both for the final assembly and for testing constructs.
- Assemble the PyrD vector
- Assemble the AmyE vector
Assembly of Vectors
Starting from the top, we are assembling the first two fragments (K14070 and K14064) and K147002 with our oligos to add in a Dif site. Two dif sites on either side of resistance cassettes can be used to later excise antibiotic resistance from our final constructs.
- K14070 and K14064 fragments
- DNA was taken out of the reigstry - Cut with restriction enzymes - Run on a gel to confirm correct cutting and estimate relative ratios of DNA for ligation - Ligated overnight - Transformed into E.Coli to Amplify the DNA - Colony PCR has been used to confirm the correct insert size.
- Next Steps:
- Extract the DNA with a miniprep - Proceed to the next step - Reverse PCR
- K14002 and oligos
- Ligated two single stranded oligos together to produce Dif and insertion site - Standard biobrick assembly of oligos to K14002 - Ligation and transformation into E.coli competent cells (strain) - Screen plated colonies for correct insertion
- Next Steps
- Purify the correct insert out of E.coli - Next step assembly - LacI test vector and Final assembly vector
- LacI Testing vector
- Currently waiting for Part B step 2 Midi-prep results to start this step
Schedule & Lab notes
PyrD Vector
Week 6
Week 6 | Monday | Tuesday | Wednesday | Thursday | Friday |
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MORNING |
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AFTERNOON |
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Week 7
Week 6 | Monday | Tuesday | Wednesday | Thursday | Friday |
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MORNING | Starting assembly of AmyE vector
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k64 and k70, k70 only, Spec and 5' PyrD diff |
Spec and 5' PyrD diff
k64 and k70. k70 only was discarded since this was purely for a baground check |
AFTERNOON |
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Week 6
Week 6 | Monday | Tuesday | Wednesday | Thursday | Friday |
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MORNING |
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AFTERNOON |
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Week 7
Week 6 | Monday | Tuesday | Wednesday | Thursday | Friday |
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MORNING | Starting assembly of AmyE vector
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k64 and k70, k70 only, Spec and 5' PyrD diff |
Spec and 5' PyrD diff
k64 and k70. k70 only was discarded since this was purely for a baground check |
AFTERNOON |
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Week 8
Week 8 | Monday | Tuesday | Wednesday | Thursday | Friday | Saturday |
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MORNING |
Set up overnight ligations for standard assembly (BBA) and 3A cloning (3A) of dif P
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AFTERNOON |
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Backbone PCR of pSB1C3 using PFU (3rd attempt)
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Week 9
Week 8 | Monday | Tuesday | Wednesday | Thursday | Friday | |
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MORNING |
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AFTERNOON |
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