IGEM:IMPERIAL/2006/project/Oscillator/project browser/Test Killing Predator Construct/Implementation: Difference between revisions
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==Ligation | ==Ligation Strategy== | ||
Step 1: Construct <bbpart>J37023</bbpart> from Part <bbpart>C0060</bbpart> and a FLAG immunotag by PCR (sequence from Dr. Mann) | Step 1: Construct <bbpart>J37023</bbpart> from Part <bbpart>C0060</bbpart> and a FLAG immunotag by PCR (sequence from Dr. Mann) | ||
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**B0014 (1G) (vector) cut with EcoRI & XbaI, expected size = 95 bp + 3189 bp (pSB1AK3) = 3284 bp | **B0014 (1G) (vector) cut with EcoRI & XbaI, expected size = 95 bp + 3189 bp (pSB1AK3) = 3284 bp | ||
*Miniprep sizes expected: 911 bp & 3189 bp (pSB1AK3) | *Miniprep sizes expected: 911 bp & 3189 bp (pSB1AK3) | ||
<u><font size="2">'''Ligating the AiiA Testing Construct:'''</font></u> | |||
__NOTOC__ | |||
{| border="1" cellpadding="0" width="100%" | |||
|- | |||
!style="background:#E6B3E0" height="25pt" width="80pt" |For !! style="background:#E6B3E0" |Stage 1 !! style="background:#E6B3E0" |Stage 2 !! style="background:#E6B3E0" |Stage 3 | |||
|- | |||
!style="background:#D9F2ED"|<center><bbpart>J37023</bbpart> <br> (PCR'd)</center> !!style="background:#CCEBAD" | <center>[[Image:J37023 logo.png]]</center> | |||
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!style="background:#D9F2ED"|<center><bbpart>J37024</bbpart>=<br>(<bbpart>J37023</bbpart>+<bbpart>B0014</bbpart>)</center>|| !!style="background:#CCEBAD" | <center>[[Image:J37023 logo.png]][[Image:Plus sign.png|20px]][[Image:B0014 logo.png]]</center> | |||
|- | |||
!style="background:#D9F2ED"|<center><bbpart>J37022</bbpart>=<br>(<bbpart>J04500</bbpart>+<bbpart>J37024</bbpart>)</center>|| || !!style="background:#CCEBAD" | <center>[[Image:J37022 logo.png]]</center> | |||
|} | |||
*More information on ligations can be found on the [http://openwetware.org/wiki/IGEM:IMPERIAL/2006/Ligation_Page Ligation Page]. | |||
==Quality control== | |||
__NOTOC__ | |||
{| border="1" cellpadding="0" width="100%" | |||
|- | |||
!height="25pt" width="80pt"| Part !! Logo !! Built !! Sequenced !! Primers Used for Sequencing | |||
|- | |||
| <center><bbpart>J37022</bbpart></center> || [[Image:J37022 logo.png]] || No || N/A || N/A | |||
|- | |||
| <center><bbpart>J37023</bbpart></center> || [[Image:J37023 logo.png]] || Yes || [http://openwetware.org/images/b/b3/J37025sequencing.doc Yes] <br> Note: This part was sequenced as a part of J37025 || [http://openwetware.org/wiki/IGEM:IMPERIAL/2006/project/parts/Primers Primers used] | |||
|- | |||
| <center><bbpart>J37024</bbpart></center> || [[Image:J37024 logo.png]] || No || N/A || N/A | |||
|} | |||
==WetLab work entries== |
Revision as of 08:02, 30 October 2006
Super Parts | Predator Construct | |||
---|---|---|---|---|
Actual Part | AiiA Testing Construct <bbpart>J37022</bbpart> | |||
Sub Parts | <bbpart>R0010</bbpart> | <bbpart>B0034</bbpart> | <bbpart>J37023</bbpart> | <bbpart>B0015</bbpart> |
Ligation Strategy
Step 1: Construct <bbpart>J37023</bbpart> from Part <bbpart>C0060</bbpart> and a FLAG immunotag by PCR (sequence from Dr. Mann)
Step 2: Construct <bbpart>J37024</bbpart> by adding a terminator (<bbpart>B0015</bbpart> to J37023
Step 3: Construct <bbpart>J37022</bbpart> by adding the LacI promoter (<bbpart>R0010</bbpart>) and the RBS <bbpart>B0034</bbpart>, which is contained within the part <bbpart>J04500</bbpart>.
People Involved with Ligations:
- John Sy
- ???
<bbpart>J37022</bbpart> (Final AiiA test construct)
- <bbpart>J04500</bbpart> (16P) + <bbpart>J37024</bbpart>
- J37024 (insert) cut with XbaI & PstI, expected size = 911 bp (on pSB1AK3 = 3189 bp)
- J04500 (16P) (vector) cut with SpeI & PstI, expected size = 220 bp + 3189 (pSB1AK3) = 3409 bp
- Miniprep sizes expected: 1131 bp & 3189 bp (pSB1AK3)
<bbpart>J37023</bbpart> (for predator cell/test construct)
- AiiA with Immunotag
- Done by PCR
- Status: Complete (awaiting sequencing results)
- Expected Part Length = 816 bp
- Expected Vector Length = Unknown (Hanging T tail vector)
<bbpart>J37024</bbpart> (for AiiA predator cell/test construct)
- <bbpart>J37023</bbpart> + <bbpart>B0015</bbpart>
- J37023 (insert) cut with EcoRI & SpeI, expected size = 816 bp
- B0014 (1G) (vector) cut with EcoRI & XbaI, expected size = 95 bp + 3189 bp (pSB1AK3) = 3284 bp
- Miniprep sizes expected: 911 bp & 3189 bp (pSB1AK3)
Ligating the AiiA Testing Construct:
- More information on ligations can be found on the Ligation Page.
Quality control
Part | Logo | Built | Sequenced | Primers Used for Sequencing |
---|---|---|---|---|
No | N/A | N/A | ||
Yes | Yes Note: This part was sequenced as a part of J37025 |
Primers used | ||
No | N/A | N/A |