Berk2006-ConjugationTeam - Revision history

Mfleming: /* SIL_07/21/06 */

2006-07-21T18:57:03Z

SIL_07/21/06

←Older revision		Revision as of 18:57, 21 July 2006
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	Also, the complements grew last night, so I mini-prepped them all today. One of the TraM complement clones came out pink which indicates it is probably the parent vector so I did not mini prep that one. I also digested them in EcoR1/Spe1 and ran them on a gel to check to see if the insert is around the right size. Also we are going to send them in for sequencing. <br>		Also, the complements grew last night, so I mini-prepped them all today. One of the TraM complement clones came out pink which indicates it is probably the parent vector so I did not mini prep that one. I also digested them in EcoR1/Spe1 and ran them on a gel to check to see if the insert is around the right size. Also we are going to send them in for sequencing. <br>
	'''Picture of complements digested in EcoR1/Spe1 (pSB1A2-J23013 (1), pSB1A2-J23013(2), pSB1A2-J23011, ladder) '''<br>		'''Picture of complements digested in EcoR1/Spe1 (pSB1A2-J23013 (1), pSB1A2-J23013(2), pSB1A2-J23011, ladder) '''<br>
-	[[Image:MF072106.jpg]]	+	[[Image:MF072106.jpg]] <br>
	We are also almost out of TriK plates and Carb plates, so Matt poured a bunch more. <br>		We are also almost out of TriK plates and Carb plates, so Matt poured a bunch more. <br>

Mfleming: /* SIL_07/21/06 */

2006-07-21T18:56:49Z

SIL_07/21/06

←Older revision		Revision as of 18:56, 21 July 2006
Line 9:		Line 9:
	Also, the complements grew last night, so I mini-prepped them all today. One of the TraM complement clones came out pink which indicates it is probably the parent vector so I did not mini prep that one. I also digested them in EcoR1/Spe1 and ran them on a gel to check to see if the insert is around the right size. Also we are going to send them in for sequencing. <br>		Also, the complements grew last night, so I mini-prepped them all today. One of the TraM complement clones came out pink which indicates it is probably the parent vector so I did not mini prep that one. I also digested them in EcoR1/Spe1 and ran them on a gel to check to see if the insert is around the right size. Also we are going to send them in for sequencing. <br>
	'''Picture of complements digested in EcoR1/Spe1 (pSB1A2-J23013 (1), pSB1A2-J23013(2), pSB1A2-J23011, ladder) '''<br>		'''Picture of complements digested in EcoR1/Spe1 (pSB1A2-J23013 (1), pSB1A2-J23013(2), pSB1A2-J23011, ladder) '''<br>
		+	[[Image:MF072106.jpg]]
	We are also almost out of TriK plates and Carb plates, so Matt poured a bunch more. <br>		We are also almost out of TriK plates and Carb plates, so Matt poured a bunch more. <br>

Mfleming: /* MF_07/21/06 */

2006-07-21T18:55:49Z

MF_07/21/06

←Older revision		Revision as of 18:55, 21 July 2006
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	[[IGEM:UC Berkeley/2006/Conjugation Research Papers\|Conjugation Research Papers]]		[[IGEM:UC Berkeley/2006/Conjugation Research Papers\|Conjugation Research Papers]]
	-----------		-----------
-	== ~~MF_07~~/21/06 ==	+	== SIL_07/21/06 ==
-		+	So, the knockout plates for the F plasmid came back as lawns that didn't even smell like e.coli (according to john) so they are definitely not what we were going for. Our guess with what went wrong is that pOX38 may be CAM resistant, which would be very bad, so now we're streaking some F plasmids, in particular Frost F, and hopefully one of them will be CAM sensitive so that we can do the knockout. <br>
		+	On the other hand, the R plasmid knockout of traG looks promising, so Matt is growing overnight cultures of 4 clones and tomorrow we will do a genomic mini-prep and map it tomorrow to see if the gene is knocked out. <br>
		+	Also, the complements grew last night, so I mini-prepped them all today. One of the TraM complement clones came out pink which indicates it is probably the parent vector so I did not mini prep that one. I also digested them in EcoR1/Spe1 and ran them on a gel to check to see if the insert is around the right size. Also we are going to send them in for sequencing. <br>
		+	'''Picture of complements digested in EcoR1/Spe1 (pSB1A2-J23013 (1), pSB1A2-J23013(2), pSB1A2-J23011, ladder) '''<br>
		+	We are also almost out of TriK plates and Carb plates, so Matt poured a bunch more. <br>

	== MF_07/20/06 ==		== MF_07/20/06 ==

Mfleming at 18:01, 21 July 2006

2006-07-21T18:01:08Z

←Older revision		Revision as of 18:01, 21 July 2006
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	\|'''WT pOX38'''\|\|+\|\|+\|\|850 \|\| n/a\|\| 1000 \|\|52		\|'''WT pOX38'''\|\|+\|\|+\|\|850 \|\| n/a\|\| 1000 \|\|52
	\|-		\|-
-	\|''' pOX 38+J01064'''\|\|+\|\|+\|\| ~~NNN~~ \|\|~~NNN~~ \|\| ?\|\| 3	+	\|''' pOX 38+DH10B/J01064'''\|\|+\|\|+\|\| 2880 (100 fold) \|\| 1760 (100 fold) \|\| 100\|\| 3
	\|-		\|-
	\|'''Olambda+1064'''\|\|+\|\|+\|\| 6 (2 red, 4 white) \|\|10 (red)\|\| 10 \|\|n/a		\|'''Olambda+1064'''\|\|+\|\|+\|\| 6 (2 red, 4 white) \|\|10 (red)\|\| 10 \|\|n/a
Line 38:		Line 38:
	\|'''TlamdaOlamda+1064+1093'''\|\|+\|\|+\|\|1\|\|0\|\| 10 \|\| n/a		\|'''TlamdaOlamda+1064+1093'''\|\|+\|\|+\|\|1\|\|0\|\| 10 \|\| n/a
	\|-		\|-
-	\|'''RP4'''\|\|+\|\|+\|\|~~NNN~~ \|\|n/a \|\| ? \|\| 38	+	\|'''RP4'''\|\|+\|\|+\|\| 30 (10 fold) \|\|n/a \|\| 10 \|\| 38
	\|-		\|-
	\|'''BW20767+JO1003'''\|\|+\|\|+\|\|n/a \|\|625 \|\| 1000 \|\| 3		\|'''BW20767+JO1003'''\|\|+\|\|+\|\|n/a \|\|625 \|\| 1000 \|\| 3
	\|-		\|-
-	\|'''Rlambda+JO1003'''\|\|+\|\|-\|\|45 \|\| 1280 \|\| ?\|\| 16	+	\|'''Rlambda+JO1003'''\|\|+\|\|-\|\|45 (1:1)\|\| 1280 (1:1) \|\| 0\|\| 16
	\|-		\|-
	\|'''Rlambda+JO1003+J01024'''\|\|+\|\|-\|\|550 \|\|0 \|\|10\|\| 2		\|'''Rlambda+JO1003+J01024'''\|\|+\|\|-\|\|550 \|\|0 \|\|10\|\| 2

Mfleming at 17:35, 21 July 2006

2006-07-21T17:35:46Z

←Older revision		Revision as of 17:35, 21 July 2006
Line 4:		Line 4:
	[[IGEM:UC Berkeley/2006/Conjugation Research Papers\|Conjugation Research Papers]]		[[IGEM:UC Berkeley/2006/Conjugation Research Papers\|Conjugation Research Papers]]
	-----------		-----------
		+	== MF_07/21/06 ==


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	\|'''BW20767+JO1003'''\|\|+\|\|+\|\|n/a \|\|625 \|\| 1000 \|\| 3		\|'''BW20767+JO1003'''\|\|+\|\|+\|\|n/a \|\|625 \|\| 1000 \|\| 3
	\|-		\|-
-	\|'''Rlambda+JO1003'''\|\|+\|\|-\|\|~~NNN~~ \|\|~~NNN~~ \|\| ?\|\| 16	+	\|'''Rlambda+JO1003'''\|\|+\|\|-\|\|45 \|\| 1280 \|\| ?\|\| 16
	\|-		\|-
	\|'''Rlambda+JO1003+J01024'''\|\|+\|\|-\|\|550 \|\|0 \|\|10\|\| 2		\|'''Rlambda+JO1003+J01024'''\|\|+\|\|-\|\|550 \|\|0 \|\|10\|\| 2

Mfleming at 17:15, 21 July 2006

2006-07-21T17:15:34Z

←Older revision		Revision as of 17:15, 21 July 2006
Line 11:		Line 11:
	[[Image:MF072006.jpg]]<br>		[[Image:MF072006.jpg]]<br>
	We also grew up overnights of the TraM and TraG biobricks (pSB1A2-J23011 and pSB1A2-J23013), so that tomorrow we can digest them in EcoR1 and Spe1 and map them to see if the fragments are around the right size, and then we will sequence them to make sure.<br>		We also grew up overnights of the TraM and TraG biobricks (pSB1A2-J23011 and pSB1A2-J23013), so that tomorrow we can digest them in EcoR1 and Spe1 and map them to see if the fragments are around the right size, and then we will sequence them to make sure.<br>
-	~~We also made -80 stocks of pKD46xpOX38 and pKD46/Rlambda. <br>~~
	Lastly, we are hopefully fininshing up the dilution series of the conjugation experiments today, we are going to do several dilution for the 3 cultures that still grew lawns or else grew nothing because of too much dilution. <br>		Lastly, we are hopefully fininshing up the dilution series of the conjugation experiments today, we are going to do several dilution for the 3 cultures that still grew lawns or else grew nothing because of too much dilution. <br>

Mfleming at 19:05, 20 July 2006

2006-07-20T19:05:32Z

←Older revision		Revision as of 19:05, 20 July 2006
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	Return to [http://www.openwetware.org/wiki/IGEM:UC_Berkeley/2006 main page]		Return to [http://www.openwetware.org/wiki/IGEM:UC_Berkeley/2006 main page]
-


	[[IGEM:UC Berkeley/2006/Conjugation Research Papers\|Conjugation Research Papers]]		[[IGEM:UC Berkeley/2006/Conjugation Research Papers\|Conjugation Research Papers]]
	-----------		-----------
		+
		+
	== MF_07/20/06 ==		== MF_07/20/06 ==
-	Today we are doing the knockouts of pKD46/pOX38 (OriT and trbC knockouts, the TraM PCR and oligos failed) and pKD46/Rlambda (traG knockout).	+	Today we are doing the knockouts of pKD46/pOX38 (OriT and trbC knockouts, the TraM PCR and oligos failed) and pKD46/Rlambda (traG knockout). We are going to electroporate the PCR products into competent cells, which we also made today.
-	Yesterday, we redid the PCR for the OriT knockout using higher concentrations of the oligos, hoping for better results and we got them this morning and the mapping came out pretty good. We are going to use the PCR from the middle lane because it came out cleaner. Here is the gel picture:<br>	+	Yesterday, we redid the PCR for the OriT knockout using higher concentrations of the oligos, hoping for better results and we got them this morning and the mapping came out pretty good. We are going to use the PCR from the middle lane because it came out cleaner. We cleaned the PCR product, digested it in dPN1 and cleaned it up again. Here is the gel picture:<br>
	[[Image:MF072006.jpg]]<br>		[[Image:MF072006.jpg]]<br>
		+	We also grew up overnights of the TraM and TraG biobricks (pSB1A2-J23011 and pSB1A2-J23013), so that tomorrow we can digest them in EcoR1 and Spe1 and map them to see if the fragments are around the right size, and then we will sequence them to make sure.<br>
		+	We also made -80 stocks of pKD46xpOX38 and pKD46/Rlambda. <br>
		+	Lastly, we are hopefully fininshing up the dilution series of the conjugation experiments today, we are going to do several dilution for the 3 cultures that still grew lawns or else grew nothing because of too much dilution. <br>
		+

	==SIL_07/19/06==		==SIL_07/19/06==

Mfleming at 18:56, 20 July 2006

2006-07-20T18:56:25Z

←Older revision		Revision as of 18:56, 20 July 2006
Line 9:		Line 9:
	Yesterday, we redid the PCR for the OriT knockout using higher concentrations of the oligos, hoping for better results and we got them this morning and the mapping came out pretty good. We are going to use the PCR from the middle lane because it came out cleaner. Here is the gel picture:<br>		Yesterday, we redid the PCR for the OriT knockout using higher concentrations of the oligos, hoping for better results and we got them this morning and the mapping came out pretty good. We are going to use the PCR from the middle lane because it came out cleaner. Here is the gel picture:<br>
	[[Image:MF072006.jpg]]<br>		[[Image:MF072006.jpg]]<br>
-	~~'''Procedure for Knockouts'''~~
-	~~#50mL LB+Carb+Kan and 5mL starter culture plus 200mg/mL Arabinose~~
-	~~#Grow to OD=.8 (the higher the better)~~
-	~~#Spin approx. 40mL in 50mL conical, 3 minutes @ 8000~~
-	~~#Resuspend 30mL 10% glycerol~~
-	~~#Spin 3 minutes @ 7500~~
-	~~#Repeat glycerol suspension (step 4)~~
-	~~#Dump supernatant, resuspend in remaining liquid (don't add anything in this step)~~
-	~~#Electroporate 50ul cells and 1ul PCR product~~
-	~~#Rescue one hour @ 37C~~
-	~~#Plate~~

	==SIL_07/19/06==		==SIL_07/19/06==

Mfleming at 18:51, 20 July 2006

2006-07-20T18:51:18Z

←Older revision		Revision as of 18:51, 20 July 2006
Line 10:		Line 10:
	[[Image:MF072006.jpg]]<br>		[[Image:MF072006.jpg]]<br>
	'''Procedure for Knockouts'''		'''Procedure for Knockouts'''
-	#50mL LB+Carb+Kan and 5mL ~~started~~ plus 200mg/mL Arabinose	+	#50mL LB+Carb+Kan and 5mL starter culture plus 200mg/mL Arabinose
	#Grow to OD=.8 (the higher the better)		#Grow to OD=.8 (the higher the better)
	#Spin approx. 40mL in 50mL conical, 3 minutes @ 8000		#Spin approx. 40mL in 50mL conical, 3 minutes @ 8000

Mfleming at 18:50, 20 July 2006

2006-07-20T18:50:10Z

←Older revision		Revision as of 18:50, 20 July 2006
Line 8:		Line 8:
	Today we are doing the knockouts of pKD46/pOX38 (OriT and trbC knockouts, the TraM PCR and oligos failed) and pKD46/Rlambda (traG knockout).		Today we are doing the knockouts of pKD46/pOX38 (OriT and trbC knockouts, the TraM PCR and oligos failed) and pKD46/Rlambda (traG knockout).
	Yesterday, we redid the PCR for the OriT knockout using higher concentrations of the oligos, hoping for better results and we got them this morning and the mapping came out pretty good. We are going to use the PCR from the middle lane because it came out cleaner. Here is the gel picture:<br>		Yesterday, we redid the PCR for the OriT knockout using higher concentrations of the oligos, hoping for better results and we got them this morning and the mapping came out pretty good. We are going to use the PCR from the middle lane because it came out cleaner. Here is the gel picture:<br>
-	[[Image:MF072006.jpg]]	+	[[Image:MF072006.jpg]]<br>
		+	'''Procedure for Knockouts'''
		+	#50mL LB+Carb+Kan and 5mL started plus 200mg/mL Arabinose
		+	#Grow to OD=.8 (the higher the better)
		+	#Spin approx. 40mL in 50mL conical, 3 minutes @ 8000
		+	#Resuspend 30mL 10% glycerol
		+	#Spin 3 minutes @ 7500
		+	#Repeat glycerol suspension (step 4)
		+	#Dump supernatant, resuspend in remaining liquid (don't add anything in this step)
		+	#Electroporate 50ul cells and 1ul PCR product
		+	#Rescue one hour @ 37C
		+	#Plate

	==SIL_07/19/06==		==SIL_07/19/06==