IGEM:Imperial/2010/Vectors team: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(→Week 7) |
|||
| Line 222: | Line 222: | ||
*<font color = #6B3FA0>Restriction digestion of '''pSB1C3''' (using Eco and Pst)</font> | *<font color = #6B3FA0>Restriction digestion of '''pSB1C3''' (using Eco and Pst)</font> | ||
*<font color = #6B3FA0>PCR purification of '''pSB1C3''' after digestion</font> | *<font color = #6B3FA0>PCR purification of '''pSB1C3''' after digestion</font> | ||
*<font color = #6B3FA0>Gel analysis of 5' ins after purification and '''pSB1C3''' to work out ratios for ligation</font> | *<font color = #6B3FA0>Gel analysis of '''5' ins''' after purification and '''pSB1C3''' to work out ratios for ligation</font> | ||
*<font color = #6B3FA0>Restriction digestion of '''pveg''' promoter and RBS [K143053] (using Eco and Spe)</font> | *<font color = #6B3FA0>Restriction digestion of '''pveg''' promoter and RBS [K143053] (using Eco and Spe)</font> | ||
| Line 231: | Line 231: | ||
*<font color = #6B3FA0>Replica plating and colony PCR of transformed colonies (containing '''5' dif in pSB1C3''')</font> | *<font color = #6B3FA0>Replica plating and colony PCR of transformed colonies (containing '''5' dif in pSB1C3''')</font> | ||
*<font color = #6B3FA0>Gel extraction and PCR purification of '''pveg''' and '''SpecR-T''' in preparation for ligations this afternoon</font> | *<font color = #6B3FA0>Gel extraction and PCR purification of '''pveg''' and '''SpecR-T''' in preparation for ligations this afternoon</font> | ||
*<font color = #6B3FA0>Gel analysis of pveg , Spec-T and pSB1C3 in order to work out ratios for the ligation</font> | *<font color = #6B3FA0>Gel analysis of '''pveg , Spec-T and pSB1C3''' in order to work out ratios for the ligation</font> | ||
|| | || | ||
*<font color = #6B3FA0>Transformation of overnight ligations: | *<font color = #6B3FA0>Transformation of overnight ligations: | ||
| Line 287: | Line 287: | ||
=====Thursday, August 19===== | =====Thursday, August 19===== | ||
=====Friday, August 20===== | =====Friday, August 20===== | ||
===Week 7=== | ===Week 7=== | ||
Revision as of 12:52, 5 September 2010
<html> <head> <style type="text/css">
- content {background-image: url(http://openwetware.org/images/2/29/Kkbgd2.JPG)}
</style>
</head>
<body>
</body>
</html>
<html> <head>
<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();
$("p[id|=popshow]").click(function() {
$("ul[id|=poplist]").slideToggle("slow");
return false;
});
});
</script>
<style type="text/css">
#popcon
{
width: 360px;
}
#popcon ul
{
margin-left: 0px;
padding-left: 0px;
list-style: none;
font-family: Arial, Helvetica, sans-serif;
}
#popshow
{
width: 360px;
margin-left: 0px;
padding-left: 0px;
list-style: none;
font-family: Arial, Helvetica, sans-serif;
display: block;
color: #fff;
text-decoration: none;
text-align: center;
background-color: #CCCCCC;
padding: 3px;
border-bottom: 1px solid #fff;
}
#popcon a:link, #popcon a:visited
{
display: block;
color: #fff;
text-decoration: none;
text-align: center;
background-color: #CCCCCC;
padding: 3px;
//border-bottom: 1px solid #FFF;
}
#popcon a:hover, #popcon a:active
{
background-color: #FFCCFF
}
</style>
</head> <body>
<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>
</ul>
</div>
</body> </html>
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 |
|---|---|---|---|---|---|
| MORNING |
| ||||
| AFTERNOON |
Start assembly of PyrD vector
|
|
Thursday, August 12
- Annealing the forward and reverse strands of the dif XP oligo
The forward and reverse strands of the 5' dif site with XbaI and PstI restriction sites on either side have been synthesized separately. The synthesized fragments arrive in solid powder form. These were immediately diluted in ddH2O to obtain a stock concentration of 1 ng/ul. They were then allowed to anneal together by heating them to 95 degs for denaturation and allowing them to cool down and anneal overnight.
Friday, August 13
- Restriction digest of dif XP
After annealing the two strands, the oligo was cut with XbaI and PstI to obtain overhangs that would later ligate with the compatible overhangs of a digested vector.
- PCR purification of the digested dif XP
The digested oligo was PCR purified in order to get rid of any contaminants. PCR purification gets rid of short pieces of DNA which are less than about 40 base pairs.
- Gel Analysis of dif XP
Week 7
| Week 6 | Monday | Tuesday | Wednesday | Thursday | Friday |
|---|---|---|---|---|---|
| MORNING |
|
|
|
5' dif with pSB1C3 and pveg, SpecR-T and pSB1C3 |
5' dif with pSB1C3 and pveg, SpecR-T and pSB1C3 |
| AFTERNOON |
|
|
|
|
Monday, August 16
- Restriction digest of 5' ins [K143008]
K143008 is the 5' integration site for the PyrD vector. This will be used as a front insert together with the dif XP for the pSB1C3 vector.
- PCR amplification of pSB1C3
The pSB1C3 vector backbone from the registry was amplified with the use of SB3 and SB2a primers. Submission of parts to the registry requires them to be in a pSB1C3 vector therefore any parts to be submitted will be inserted into this vector.
- PCR purification of pSB1C3
The PCR amplified vector was purified in order to get rid of any contaminants. For example, short pieces of DNA like the primers.
Tuesday, August 17
- Gel extraction and purification of 5' ins ES
The digested 5' ins was first analyzed on the gel to verify it's size and then extracted for purification. The 5' ins was gel purified in order to extract only the relevant piece of DNA.
- Restriction digest of pSB1C3
The pSB1C3 vector was digested so that it would contain compatible overhangs for ligation with inserts.
- PCR purification of pSB1C3 EP
The digested pSB1C3 was re-purified in order to get rid of any contaminant DNA that arose during the digestion.
- Restriction digest of pveg
Wednesday, August 18
Thursday, August 19
Friday, August 20
Week 7
| Week 6 | Monday | Tuesday | Wednesday | Thursday | Friday |
|---|---|---|---|---|---|
| MORNING | Starting assembly of AmyE vector
|
|
|
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 |
|
|
|
|
Week 8
| Week 8 | Monday | Tuesday | Wednesday | Thursday | Friday | Saturday |
|---|---|---|---|---|---|---|
| MORNING |
Set up overnight ligations for standard assembly (BBA) and 3A cloning (3A) of dif P
|
|
|
|
|
|
| AFTERNOON |
|
|
|
|
Backbone PCR of pSB1C3 using PFU (3rd attempt)
|
|
Week 9
| Week 8 | Monday | Tuesday | Wednesday | Thursday | Friday | |
|---|---|---|---|---|---|---|
| MORNING |
|
|
|
|
|
|
| AFTERNOON |
|
|
|
|
|
