Team:Amsterdam/extra/diary

From 2012.igem.org

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<h1>Diary</h1>
<h1>Diary</h1>
-
11/05/2012
+
12/07/2012
-
Gel electrophoresis of the PCR reaction performed on 10/05/2012. Non-specific products obtained. Repeat PCR? Change annealing temperature?
+
Transformation of the plasmid containing the M.ScaI methyltransferase (pIDTSMART-AMP).
-
Ladder
+
11/07/2012
-
pET28a+ (1 ng for PCR)
+
Mini-prep BBa_J63009+BBa_J04450 and pSB1AT3+BBa_J45320. Elution in 25µl.
-
pET28a+ (10 ng for PCR)
+
Restriction digestion of BBa_J63009+BBa_J04450 with DraI and RsaI/XbaI.
-
peGFP (1 ng for PCR)
+
Restriction digestion of pSB1AT3+BBa_J45320 with ScaI and xhoI.
-
peGFP (10 ng for PCR)
+
Something went wrong with the digestions.....
 +
Transformation:
 +
1)pSB3C5+ BBa_J04450 (2012 iGEM distribution kit plate 1 Well 3C)
 +
2)pSB4C5+ BBa_J04450 (2012 iGEM distribution kit plate 1 Well 3E)
-
Second PCR for peGFP and pET28a+. annealing temperature = 53˚C
+
10/07/2012
-
Same results obtained..... :(
+
Set overnight cultures for BBa_J63009+BBa_J04450 and pSB1AT3+BBa_J45320 for mini-prep.
-
14/05/2012
+
09/07/2012
-
PCR of pET28a+ and peGFP -> annealing temperature 56˚C. non-specific DNA products obtained for the vector. Increase annealing temperature? Gel extract?
+
Symposium with all the dutch iGEM teams at Wageningen University.
 +
No plates for pSB1C3 again.... Noooooooooo!
-
Ladder
+
06/07/2012
-
Original pT28a+
+
Re-transform pSB1C3!
-
Transformed pET28a+ (200 pg DNA for PCR)
+
The overnight culture for BBa_J63009+BBa_J04450 and pSB1AT3+BBa_J45320 turned pink! What is going on?
-
Transformed pET28a+ (1ng DNA for PCR)
+
At the end of the day -> BBa_J63009+BBa_J04450 and pSB1AT3+BBa_J45320 both contain RFP......the colonies should be reddish..... Oops....already threw them away. Awwwww start again then!
-
Original peGFP
+
-
Transformed peGFP (200 pg DNA for PCR)
+
-
Transformed peGFP (1 ng DNA for PCR)
+
-
Gel extraction of pET28a+ from TAE gel + ethidium bromide. Elution in 25 µl miliQ water.
+
05/07/2012
-
PCR No. 2 -> pET28a+ and peGFP -> annealing temperature 60˚C.
+
Isolated colonies obtained for BBa_J63009+BBa_J04450 and pSB1AT3+BBa_J45320.
 +
Set up overnight liquid LB culture for each plasmid for mini-prep.
-
16/05/2012
+
04/07/2012
 +
Plates show growth. Antibiotics are working! Swarming of bacteria. Unusable!
 +
Streak new plates with a single colony to obtain isolated colonies. Incubate overnight at 37˚C.
 +
Nothing grew on the pSB1C3 plate.
 +
 
 +
03/07/2012
 +
Prepared ampicillin and chloramphenicol stock solutions (50 mg/ml).
 +
Made LB/ampicillin and LB/chloramphenicol plates.
 +
Transformation:
 +
1)pSB1AT3+BBa_J45320 (2012 iGEM distribution kit plate 2 Well 5J)
 +
2)BBa_J63009+BBa_J04450 (2012 iGEM distribution kit plate 1 Well 1A)
 +
3)pSB1C3 (2012 iGEM distribution kit linearised plasmid)
 +
Incubate overnight at 37˚C.06/06/2012
 +
Mini-prep of the colony obtained from Gibson on 05/06. Elution in 25µl miliQ water.
 +
Digestion of extracted plasmid with NotI and BglII (double digest).
 +
 
 +
PICTUREEEEEEE
-
pET28a+ (gel extracted)
 
-
peGFP (gel extracted)
 
Ladder
Ladder
-
pET28a+ (PCR No.2 -> 14/05/2012)
+
Mini-prep colony digested with NotI and BglII
-
peGFP (PCR No.2 -> 14/05/2012)
+
-
Still non-specific products observed for the PCR. Possible inter-change during gel extraction; pET28a+ and peGFP got mixed up?
+
05/06/2012
 +
One colony obtained. Checked for fluorescence with GFP. No fluorescence.
 +
Prepared overnight culture with colony for mini-prep.
-
23/05/2012
+
01/06/2012
-
Repeat what was done on 14/05 (PCR Temp 60˚C + gel extraction.
+
Diewertje removed the plates from the incubator and put them at 4˚C for the weekend. Thank you Diewertje!
-
Two products obtained and concentration seems too low. Pfffffff.... Repeat again......And this is only a test, hope it will work better for the actual experiments
+
31/05/2012
-
:(
+
Prepared LB agar for LB plates + Kanamycin.
 +
Gibson assembly of peGFP and pET28a+ in equimolar amounts.
 +
Transformation of 5µl of Gibson reaction mix in DH5 alpha competent bacteria. Incubation overnight at 37˚C.
 +
30/05/2012
 +
Prepared TAE and TBE gels with ethidium bromide.
 +
Pooled all the PCR products for peGFP and gel extract the correct band. Elution in 25µl miliQ water.
-
24/05/2012
+
PICTUUUUREEEE
-
PCR pET28a+ -> annealing temperature 62˚C.
+
Ladder
Ladder
-
pET28a+
+
peGFP after gel extraction
-
 
+
Prepared the master mix for the Gibson assembly. Aliquoted 15µl in eppendorfs.
-
25/05/2012
+
Everything is ready! :)
-
Pooled all the PCR strips containing pET28a+ for DNA precipitation reaction.
+
29/05/2012
29/05/2012
Gel electrophoresis of precipitated DNA pET28a+ from 25/05/2012
Gel electrophoresis of precipitated DNA pET28a+ from 25/05/2012
 +
 +
PICTUUUURE
Ladder
Ladder
Line 69: Line 91:
The pET28a+ is ready for the one step isothermal gibson assembly reaction. peGFP should also be gel extracted.
The pET28a+ is ready for the one step isothermal gibson assembly reaction. peGFP should also be gel extracted.
 +
25/05/2012
 +
Pooled all the PCR strips containing pET28a+ for DNA precipitation reaction.
-
30/05/2012
+
24/05/2012
-
Prepared TAE and TBE gels with ethidium bromide.
+
PCR pET28a+ -> annealing temperature 62˚C.
-
Pooled all the PCR products for peGFP and gel extract the correct band. Elution in 25µl miliQ water.
+
-
 
+
 +
PICTUUUUUREEE
Ladder
Ladder
-
peGFP after gel extraction
+
pET28a+
-
Prepared the master mix for the Gibson assembly. Aliquoted 15µl in eppendorfs.
+
23/05/2012
-
Everything is ready! :)
+
Repeat what was done on 14/05 (PCR Temp 60˚C + gel extraction.
-
31/05/2012
+
PICTUUUUURE
-
Prepared LB agar for LB plates + Kanamycin.
+
-
Gibson assembly of peGFP and pET28a+ in equimolar amounts.
+
-
Transformation of 5µl of Gibson reaction mix in DH5 alpha competent bacteria. Incubation overnight at 37˚C.
+
-
01/06/2012
+
Two products obtained and concentration seems too low. Pfffffff.... Repeat again......And this is only a test, hope it will work better for the actual experiments
-
Diewertje removed the plates from the incubator and put them at 4˚C for the weekend. Thank you Diewertje!
+
:(
 +
16/05/2012
-
05/06/2012
+
PICTUUUURE
-
One colony obtained. Checked for fluorescence with GFP. No fluorescence.
+
-
Prepared overnight culture with colony for mini-prep.
+
-
 
+
-
06/06/2012
+
-
Mini-prep of the colony obtained from Gibson on 05/06. Elution in 25µl miliQ water.
+
-
Digestion of extracted plasmid with NotI and BglII (double digest).
+
 +
pET28a+ (gel extracted)
 +
peGFP (gel extracted)
Ladder
Ladder
-
Mini-prep colony digested with NotI and BglII
+
pET28a+ (PCR No.2 -> 14/05/2012)
 +
peGFP (PCR No.2 -> 14/05/2012)
-
03/07/2012
+
Still non-specific products observed for the PCR. Possible inter-change during gel extraction; pET28a+ and peGFP got mixed up?
-
Prepared ampicillin and chloramphenicol stock solutions (50 mg/ml).
+
-
Made LB/ampicillin and LB/chloramphenicol plates.
+
-
Transformation:
+
-
1)pSB1AT3+BBa_J45320 (2012 iGEM distribution kit plate 2 Well 5J)
+
-
2)BBa_J63009+BBa_J04450 (2012 iGEM distribution kit plate 1 Well 1A)
+
-
3)pSB1C3 (2012 iGEM distribution kit linearised plasmid)
+
-
Incubate overnight at 37˚C.
+
-
04/07/2012
+
14/05/2012
-
Plates show growth. Antibiotics are working! Swarming of bacteria. Unusable!
+
PCR of pET28a+ and peGFP -> annealing temperature 56˚C. non-specific DNA products obtained for the vector. Increase annealing temperature? Gel extract?
-
Streak new plates with a single colony to obtain isolated colonies. Incubate overnight at 37˚C.
+
-
Nothing grew on the pSB1C3 plate.
+
-
05/07/2012
+
PIIICTUUUUUREEEE
-
Isolated colonies obtained for BBa_J63009+BBa_J04450 and pSB1AT3+BBa_J45320.
+
-
Set up overnight liquid LB culture for each plasmid for mini-prep.
+
-
06/07/2012
+
Ladder
-
Re-transform pSB1C3!
+
Original pT28a+
-
The overnight culture for BBa_J63009+BBa_J04450 and pSB1AT3+BBa_J45320 turned pink! What is going on?
+
Transformed pET28a+ (200 pg DNA for PCR)
-
At the end of the day -> BBa_J63009+BBa_J04450 and pSB1AT3+BBa_J45320 both contain RFP......the colonies should be reddish..... Oops....already threw them away. Awwwww start again then!
+
Transformed pET28a+ (1ng DNA for PCR)
 +
Original peGFP
 +
Transformed peGFP (200 pg DNA for PCR)
 +
Transformed peGFP (1 ng DNA for PCR)
-
09/07/2012
+
Gel extraction of pET28a+ from TAE gel + ethidium bromide. Elution in 25 µl miliQ water.
-
Symposium with all the dutch iGEM teams at Wageningen University.
+
PCR No. 2 -> pET28a+ and peGFP -> annealing temperature 60˚C.
-
No plates for pSB1C3 again.... Noooooooooo!
+
-
10/07/2012
+
11/05/2012
-
Set overnight cultures for BBa_J63009+BBa_J04450 and pSB1AT3+BBa_J45320 for mini-prep.
+
Gel electrophoresis of the PCR reaction performed on 10/05/2012. Non-specific products obtained. Repeat PCR? Change annealing temperature?
-
11/07/2012
+
PICTUUUUUUREEEE
-
Mini-prep BBa_J63009+BBa_J04450 and pSB1AT3+BBa_J45320. Elution in 25µl.
+
-
Restriction digestion of BBa_J63009+BBa_J04450 with DraI and RsaI/XbaI.
+
-
Restriction digestion of pSB1AT3+BBa_J45320 with ScaI and xhoI.
+
-
Something went wrong with the digestions.....
+
-
Transformation:
+
-
1)pSB3C5+ BBa_J04450 (2012 iGEM distribution kit plate 1 Well 3C)
+
-
2)pSB4C5+ BBa_J04450 (2012 iGEM distribution kit plate 1 Well 3E)
+
-
12/07/2012
+
Ladder
-
Transformation of the plasmid containing the M.ScaI methyltransferase (pIDTSMART-AMP).
+
pET28a+ (1 ng for PCR)
 +
pET28a+ (10 ng for PCR)
 +
peGFP (1 ng for PCR)
 +
peGFP (10 ng for PCR)
 +
 
 +
Second PCR for peGFP and pET28a+. annealing temperature = 53˚C
 +
Same results obtained..... :(
</div>
</div>
</div>
</div>
{{Team:Amsterdam/Foot}}
{{Team:Amsterdam/Foot}}

Revision as of 11:04, 26 September 2012

Diary

12/07/2012 Transformation of the plasmid containing the M.ScaI methyltransferase (pIDTSMART-AMP).

11/07/2012 Mini-prep BBa_J63009+BBa_J04450 and pSB1AT3+BBa_J45320. Elution in 25µl. Restriction digestion of BBa_J63009+BBa_J04450 with DraI and RsaI/XbaI. Restriction digestion of pSB1AT3+BBa_J45320 with ScaI and xhoI. Something went wrong with the digestions..... Transformation: 1)pSB3C5+ BBa_J04450 (2012 iGEM distribution kit plate 1 Well 3C) 2)pSB4C5+ BBa_J04450 (2012 iGEM distribution kit plate 1 Well 3E)

10/07/2012 Set overnight cultures for BBa_J63009+BBa_J04450 and pSB1AT3+BBa_J45320 for mini-prep.

09/07/2012 Symposium with all the dutch iGEM teams at Wageningen University. No plates for pSB1C3 again.... Noooooooooo!

06/07/2012 Re-transform pSB1C3! The overnight culture for BBa_J63009+BBa_J04450 and pSB1AT3+BBa_J45320 turned pink! What is going on? At the end of the day -> BBa_J63009+BBa_J04450 and pSB1AT3+BBa_J45320 both contain RFP......the colonies should be reddish..... Oops....already threw them away. Awwwww start again then!

05/07/2012 Isolated colonies obtained for BBa_J63009+BBa_J04450 and pSB1AT3+BBa_J45320. Set up overnight liquid LB culture for each plasmid for mini-prep.

04/07/2012 Plates show growth. Antibiotics are working! Swarming of bacteria. Unusable! Streak new plates with a single colony to obtain isolated colonies. Incubate overnight at 37˚C. Nothing grew on the pSB1C3 plate.

03/07/2012 Prepared ampicillin and chloramphenicol stock solutions (50 mg/ml). Made LB/ampicillin and LB/chloramphenicol plates. Transformation: 1)pSB1AT3+BBa_J45320 (2012 iGEM distribution kit plate 2 Well 5J) 2)BBa_J63009+BBa_J04450 (2012 iGEM distribution kit plate 1 Well 1A) 3)pSB1C3 (2012 iGEM distribution kit linearised plasmid) Incubate overnight at 37˚C.06/06/2012 Mini-prep of the colony obtained from Gibson on 05/06. Elution in 25µl miliQ water. Digestion of extracted plasmid with NotI and BglII (double digest).

PICTUREEEEEEE

Ladder Mini-prep colony digested with NotI and BglII

05/06/2012 One colony obtained. Checked for fluorescence with GFP. No fluorescence. Prepared overnight culture with colony for mini-prep.

01/06/2012 Diewertje removed the plates from the incubator and put them at 4˚C for the weekend. Thank you Diewertje!

31/05/2012 Prepared LB agar for LB plates + Kanamycin. Gibson assembly of peGFP and pET28a+ in equimolar amounts. Transformation of 5µl of Gibson reaction mix in DH5 alpha competent bacteria. Incubation overnight at 37˚C.

30/05/2012 Prepared TAE and TBE gels with ethidium bromide. Pooled all the PCR products for peGFP and gel extract the correct band. Elution in 25µl miliQ water.

PICTUUUUREEEE

Ladder peGFP after gel extraction

Prepared the master mix for the Gibson assembly. Aliquoted 15µl in eppendorfs. Everything is ready! :)

29/05/2012 Gel electrophoresis of precipitated DNA pET28a+ from 25/05/2012

PICTUUUURE

Ladder Gel extracted pET28a+ peGFP from 23/05

The pET28a+ is ready for the one step isothermal gibson assembly reaction. peGFP should also be gel extracted.

25/05/2012 Pooled all the PCR strips containing pET28a+ for DNA precipitation reaction.

24/05/2012 PCR pET28a+ -> annealing temperature 62˚C.

PICTUUUUUREEE

Ladder pET28a+

23/05/2012 Repeat what was done on 14/05 (PCR Temp 60˚C + gel extraction.

PICTUUUUURE

Two products obtained and concentration seems too low. Pfffffff.... Repeat again......And this is only a test, hope it will work better for the actual experiments

(

16/05/2012

PICTUUUURE

pET28a+ (gel extracted) peGFP (gel extracted) Ladder pET28a+ (PCR No.2 -> 14/05/2012) peGFP (PCR No.2 -> 14/05/2012)

Still non-specific products observed for the PCR. Possible inter-change during gel extraction; pET28a+ and peGFP got mixed up?

14/05/2012 PCR of pET28a+ and peGFP -> annealing temperature 56˚C. non-specific DNA products obtained for the vector. Increase annealing temperature? Gel extract?

PIIICTUUUUUREEEE

Ladder Original pT28a+ Transformed pET28a+ (200 pg DNA for PCR) Transformed pET28a+ (1ng DNA for PCR) Original peGFP Transformed peGFP (200 pg DNA for PCR) Transformed peGFP (1 ng DNA for PCR)

Gel extraction of pET28a+ from TAE gel + ethidium bromide. Elution in 25 µl miliQ water. PCR No. 2 -> pET28a+ and peGFP -> annealing temperature 60˚C.

11/05/2012 Gel electrophoresis of the PCR reaction performed on 10/05/2012. Non-specific products obtained. Repeat PCR? Change annealing temperature?

PICTUUUUUUREEEE

Ladder pET28a+ (1 ng for PCR) pET28a+ (10 ng for PCR) peGFP (1 ng for PCR) peGFP (10 ng for PCR)

Second PCR for peGFP and pET28a+. annealing temperature = 53˚C Same results obtained..... :(

Retrieved from "http://2012.igem.org/Team:Amsterdam/extra/diary"