Edinburgh/1 July 2008

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Edinburgh iGEM 2008

June
M	T	W	T	F	S	S
						[http://2008.igem.org/wiki/index.php?title=Edinburgh/1_June_2008&action=edit 1]
[http://2008.igem.org/wiki/index.php?title=Edinburgh/2_June_2008&action=edit 2]	[http://2008.igem.org/wiki/index.php?title=Edinburgh/3_June_2008&action=edit 3]	[http://2008.igem.org/wiki/index.php?title=Edinburgh/4_June_2008&action=edit 4]	[http://2008.igem.org/wiki/index.php?title=Edinburgh/5_June_2008&action=edit 5]	[http://2008.igem.org/wiki/index.php?title=Edinburgh/6_June_2008&action=edit 6]	[http://2008.igem.org/wiki/index.php?title=Edinburgh/7_June_2008&action=edit 7]	[http://2008.igem.org/wiki/index.php?title=Edinburgh/8_June_2008&action=edit 8]
[http://2008.igem.org/wiki/index.php?title=Edinburgh/9_June_2008&action=edit 9]	[http://2008.igem.org/wiki/index.php?title=Edinburgh/10_June_2008&action=edit 10]	[http://2008.igem.org/wiki/index.php?title=Edinburgh/11_June_2008&action=edit 11]	[http://2008.igem.org/wiki/index.php?title=Edinburgh/12_June_2008&action=edit 12]	[http://2008.igem.org/wiki/index.php?title=Edinburgh/13_June_2008&action=edit 13]	[http://2008.igem.org/wiki/index.php?title=Edinburgh/14_June_2008&action=edit 14]	[http://2008.igem.org/wiki/index.php?title=Edinburgh/15_June_2008&action=edit 15]
[http://2008.igem.org/wiki/index.php?title=Edinburgh/16_June_2008&action=edit 16]	[http://2008.igem.org/Edinburgh/17_June_2008 17]	[http://2008.igem.org/wiki/index.php?title=Edinburgh/18_June_2008&action=edit 18]	[http://2008.igem.org/Edinburgh/19_June_2008 19]	[http://2008.igem.org/Edinburgh/20_June_2008 20]	[http://2008.igem.org/wiki/index.php?title=Edinburgh/21_June_2008&action=edit 21]	[http://2008.igem.org/wiki/index.php?title=Edinburgh/22_June_2008&action=edit 22]
[http://2008.igem.org/wiki/index.php?title=Edinburgh/23_June_2008&action=edit 23]	[http://2008.igem.org/Edinburgh/24_June_2008 24]	[http://2008.igem.org/Edinburgh/25_June_2008 25]	[http://2008.igem.org/Edinburgh/26_June_2008 26]	[http://2008.igem.org/Edinburgh/27_June_2008 27]	[http://2008.igem.org/Edinburgh/28_June_2008 28]	[http://2008.igem.org/Edinburgh/29_June_2008 29]
[http://2008.igem.org/Edinburgh/30_June_2008 30]

July
M	T	W	T	F	S	S
	[http://2008.igem.org/Edinburgh/1_July_2008 1]	[http://2008.igem.org/Edinburgh/2_July_2008 2]	[http://2008.igem.org/Edinburgh/3_July_2008 3]	[http://2008.igem.org/Edinburgh/4_July_2008 4]	[http://2008.igem.org/Edinburgh/5_July_2008 5]	[http://2008.igem.org/Edinburgh/6_July_2008 6]
[http://2008.igem.org/Edinburgh/7_July_2008 7]	[http://2008.igem.org/Edinburgh/8_July_2008 8]	[http://2008.igem.org/Edinburgh/9_July_2008 9]	[http://2008.igem.org/Edinburgh/10_July_2008 10]	[http://2008.igem.org/Edinburgh/11_July_2008 11]	[http://2008.igem.org/Edinburgh/12_July_2008 12]	[http://2008.igem.org/Edinburgh/13_July_2008 13]
[http://2008.igem.org/Edinburgh/14_July_2008 14]	[http://2008.igem.org/Edinburgh/15_July_2008 15]	[http://2008.igem.org/Edinburgh/16_July_2008 16]	[http://2008.igem.org/Edinburgh/17_July_2008 17]	[http://2008.igem.org/Edinburgh/18_July_2008 18]	[http://2008.igem.org/Edinburgh/19_July_2008 19]	[http://2008.igem.org/Edinburgh/20_July_2008 20]
[http://2008.igem.org/Edinburgh/21_July_2008 21]	[http://2008.igem.org/Edinburgh/22_July_2008 22]	[http://2008.igem.org/Edinburgh/23_July_2008 23]	[http://2008.igem.org/Edinburgh/24_July_2008 24]	[http://2008.igem.org/Edinburgh/25_July_2008 25]	[http://2008.igem.org/Edinburgh/26_July_2008 26]	[http://2008.igem.org/Edinburgh/27_July_2008 27]
[http://2008.igem.org/Edinburgh/28_July_2008 28]	[http://2008.igem.org/Edinburgh/29_July_2008 29]	[http://2008.igem.org/Edinburgh/30_July_2008 30]	[http://2008.igem.org/Edinburgh/31_July_2008 31]

August
M	T	W	T	F	S	S
				[http://2008.igem.org/Edinburgh/1_August_2008 1]	[http://2008.igem.org/wiki/index.php?title=Edinburgh/2_August_2008&action=edit 2]	[http://2008.igem.org/Edinburgh/3_August_2008 3]
[http://2008.igem.org/Edinburgh/4_August_2008 4]	[http://2008.igem.org/Edinburgh/5_August_2008 5]	[http://2008.igem.org/Edinburgh/6_August_2008 6]	[http://2008.igem.org/Edinburgh/7_August_2008 7]	[http://2008.igem.org/Edinburgh/8_August_2008 8]	[http://2008.igem.org/Edinburgh/9_August_2008 9]	[http://2008.igem.org/Edinburgh/10_August_2008 10]
[http://2008.igem.org/Edinburgh/11_August_2008 11]	[http://2008.igem.org/Edinburgh/12_August_2008 12]	[http://2008.igem.org/Edinburgh/13_August_2008 13]	[http://2008.igem.org/Edinburgh/14_August_2008 14]	[http://2008.igem.org/Edinburgh/15_August_2008 15]	[http://2008.igem.org/Edinburgh/16_August_2008 16]	[http://2008.igem.org/Edinburgh/17_August_2008 17]
[http://2008.igem.org/Edinburgh/18_August_2008 18]	[http://2008.igem.org/Edinburgh/19_August_2008 19]	[http://2008.igem.org/Edinburgh/20_August_2008 20]	[http://2008.igem.org/Edinburgh/21_August_2008 21]	[http://2008.igem.org/Edinburgh/22_August_2008 22]	[http://2008.igem.org/wiki/index.php?title=Edinburgh/23_August_2008&action=edit 23]	[http://2008.igem.org/wiki/index.php?title=Edinburgh/24_August_2008&action=edit 24]
[http://2008.igem.org/Edinburgh/25_August_2008 25]	[http://2008.igem.org/Edinburgh/26_August_2008 26]	[http://2008.igem.org/Edinburgh/27_August_2008 27]	[http://2008.igem.org/Edinburgh/28_August_2008 28]	[http://2008.igem.org/Edinburgh/29_August_2008 29]	[http://2008.igem.org/wiki/index.php?title=Edinburgh/30_August_2008&action=edit 30]	[http://2008.igem.org/wiki/index.php?title=Edinburgh/31_August_2008&action=edit 31]

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Week 3

Tuesday 1 July 08

Gel 3: minipreps M2, M3, M4 and M6 (pSB1A2+dxs clones) digested with EcoRI alone; lanes 5 and 6, 2.5μl of ligations L1 and L2. Result: all 4 minipreps now give a 4.2kb band (plus the same 3.2kb 'ghost' band as before) consistent with pSB1A2 carrying a 2kb insert. Would be nice to confirm identity using HindIII (2 internal sites). Both ligations show clear signs of insert and vector bands (oddly, the dxs insert band overlaps the pSB1A2 vector band whereas the appY insert band overlaps the lacZ insert excised from Edinbrick1). However, no ligated bands are visible. Thus the DNA purification is fine; if there was a a problem, it is with the ligase or ligase buffer.
Gel 4: Minipreps M7 to M12 (supposed to be glgC in Babel vectors) digested with EcoRI and PstI to excise the inserts. M10 and M11 have a single 3kb band consistent with vector, but no insert band at 1.2kb. M7, M8, M9 and M12 all show a single band at about 2.4kb which is not consistent with Babel vectors if properly digested. Unless the digests totally failed, none of these plasmids would seem to contain glgC.
- BUT WAIT: Turns out gel 4 has undigested plasmid DNA rather than the EcoRI/PstI digests. These were run on Gel 5. Since glgC is about 1295bp with internal EcoRI sites at +570 and +1067, we would expect to see a vector band at 3kb and the insert cut into bands of around 570bp, 567bp and 228bp. This is precisely what we see in the case of M10 and M11. We can therefore conclude that M10 and M11 both represent the Babel2+glgC BioBrick. This is not sufficient to determine the orientation, but a single EcoRI digest of these two clones should be sufficient to establish that. Even if both are in the reverse orientation, they can still be used as templates for the three mutagenesis step which will be required to produce the final BioBrick.
Also attempted HindIII digests of M2, M3, M4 and M6 to confirm that the insert is really dxs, despite knowing that the HindIII stock expired in 2002. Results are shown on Gel 6 lanes 1 to 4. In all cases a 0.6kb band was excised, consistent with dxs insert. It therefore seems likely that M2, M3, M4 and M6 all represent pSB1A2+dxs coding sequence BioBrick.
Also did fusion PCR for the BABEL1+glgC and BABEL2+glgC constructs, using 1μl of L3 and L4 as templates (P4 and P5). KOD was used, with primers stdvectf1 and glgCr1, annealing at 65°C and extending for 110s. Results are shown on Gel 6 lanes 5 and 6. P4 (BABEL1) has a strong band the right size and would probably work if self-ligated, but P5 (BABEL2) shows strong bands at 2 and 3 kb, as CK is also seeing. I suspect that primer stdvectf1 does not work well with BABEL2 - possibly mis-anneals to the other end of the vector. In any event, we can delay the decision as to whether or not to carry on with these until we know the results from M10 and M11.

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