Team:Amsterdam/extra/diary

From 2012.igem.org

(Difference between revisions)
Line 7: Line 7:
<div id="sub-menu" class="content-block">
<div id="sub-menu" class="content-block">
<h1>Diary</h1>
<h1>Diary</h1>
-
12/07/2012
+
<h4>12/07/2012</h4>
Transformation of the plasmid containing the M.ScaI methyltransferase (pIDTSMART-AMP).
Transformation of the plasmid containing the M.ScaI methyltransferase (pIDTSMART-AMP).
-
11/07/2012
+
<h4>11/07/2012</h4>
Mini-prep BBa_J63009+BBa_J04450 and pSB1AT3+BBa_J45320. Elution in 25µl.
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 BBa_J63009+BBa_J04450 with DraI and RsaI/XbaI.
Restriction digestion of pSB1AT3+BBa_J45320 with ScaI and xhoI.
Restriction digestion of pSB1AT3+BBa_J45320 with ScaI and xhoI.
 +
Something went wrong with the digestions.....
Something went wrong with the digestions.....
 +
Transformation:  
Transformation:  
1)pSB3C5+ BBa_J04450 (2012 iGEM distribution kit plate 1 Well 3C)
1)pSB3C5+ BBa_J04450 (2012 iGEM distribution kit plate 1 Well 3C)
2)pSB4C5+ BBa_J04450 (2012 iGEM distribution kit plate 1 Well 3E)
2)pSB4C5+ BBa_J04450 (2012 iGEM distribution kit plate 1 Well 3E)
-
10/07/2012
+
<h4>10/07/2012</h4>
Set overnight cultures for BBa_J63009+BBa_J04450 and pSB1AT3+BBa_J45320 for mini-prep.
Set overnight cultures for BBa_J63009+BBa_J04450 and pSB1AT3+BBa_J45320 for mini-prep.
-
09/07/2012
+
<h4>09/07/2012</h4>
Symposium with all the dutch iGEM teams at Wageningen University.
Symposium with all the dutch iGEM teams at Wageningen University.
 +
No plates for pSB1C3 again.... Noooooooooo!
No plates for pSB1C3 again.... Noooooooooo!
-
06/07/2012
+
<h4>06/07/2012</h4>
Re-transform pSB1C3!
Re-transform pSB1C3!
 +
The overnight culture for BBa_J63009+BBa_J04450 and pSB1AT3+BBa_J45320 turned pink! What is going on?
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!
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
+
<h4>05/07/2012</h4>
Isolated colonies obtained for BBa_J63009+BBa_J04450 and pSB1AT3+BBa_J45320.
Isolated colonies obtained for BBa_J63009+BBa_J04450 and pSB1AT3+BBa_J45320.
 +
Set up overnight liquid LB culture for each plasmid for mini-prep.
Set up overnight liquid LB culture for each plasmid for mini-prep.
-
04/07/2012
+
<h4>04/07/2012</h4>
Plates show growth. Antibiotics are working! Swarming of bacteria. Unusable!
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.
Streak new plates with a single colony to obtain isolated colonies. Incubate overnight at 37˚C.
 +
Nothing grew on the pSB1C3 plate.
Nothing grew on the pSB1C3 plate.
-
03/07/2012
+
<h4>03/07/2012</h4>
Prepared ampicillin and chloramphenicol stock solutions (50 mg/ml).
Prepared ampicillin and chloramphenicol stock solutions (50 mg/ml).
 +
Made LB/ampicillin and LB/chloramphenicol plates.
Made LB/ampicillin and LB/chloramphenicol plates.
 +
Transformation:  
Transformation:  
-
1)pSB1AT3+BBa_J45320 (2012 iGEM distribution kit plate 2 Well 5J)
+
*pSB1AT3+BBa_J45320 (2012 iGEM distribution kit plate 2 Well 5J)
-
2)BBa_J63009+BBa_J04450 (2012 iGEM distribution kit plate 1 Well 1A)
+
*BBa_J63009+BBa_J04450 (2012 iGEM distribution kit plate 1 Well 1A)
-
3)pSB1C3 (2012 iGEM distribution kit linearised plasmid)
+
*pSB1C3 (2012 iGEM distribution kit linearised plasmid)
Incubate overnight at 37˚C.06/06/2012
Incubate overnight at 37˚C.06/06/2012
Mini-prep of the colony obtained from Gibson on 05/06. Elution in 25µl miliQ water.
Mini-prep of the colony obtained from Gibson on 05/06. Elution in 25µl miliQ water.
Line 56: Line 66:
Mini-prep colony digested with NotI and BglII
Mini-prep colony digested with NotI and BglII
-
05/06/2012
+
<h4>05/06/2012</h4>
One colony obtained. Checked for fluorescence with GFP. No fluorescence.
One colony obtained. Checked for fluorescence with GFP. No fluorescence.
Prepared overnight culture with colony for mini-prep.
Prepared overnight culture with colony for mini-prep.
-
01/06/2012
+
<h4>01/06/2012</h4>
Diewertje removed the plates from the incubator and put them at 4˚C for the weekend. Thank you Diewertje!
Diewertje removed the plates from the incubator and put them at 4˚C for the weekend. Thank you Diewertje!
-
31/05/2012
+
<h4>31/05/2012</h4>
Prepared LB agar for LB plates + Kanamycin.
Prepared LB agar for LB plates + Kanamycin.
Gibson assembly of peGFP and pET28a+ in equimolar amounts.
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.
Transformation of 5µl of Gibson reaction mix in DH5 alpha competent bacteria. Incubation overnight at 37˚C.
-
30/05/2012
+
<h4>30/05/2012</h4>
Prepared TAE and TBE gels with ethidium bromide.
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.
Pooled all the PCR products for peGFP and gel extract the correct band. Elution in 25µl miliQ water.
Line 80: Line 90:
Everything is ready! :)
Everything is ready! :)
-
29/05/2012
+
<h4>29/05/2012</h4>
Gel electrophoresis of precipitated DNA pET28a+ from 25/05/2012
Gel electrophoresis of precipitated DNA pET28a+ from 25/05/2012
Line 91: Line 101:
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
+
<h4>25/05/2012</h4>
Pooled all the PCR strips containing pET28a+ for DNA precipitation reaction.
Pooled all the PCR strips containing pET28a+ for DNA precipitation reaction.
-
24/05/2012
+
<h4>24/05/2012</h4>
PCR pET28a+ -> annealing temperature 62˚C.
PCR pET28a+ -> annealing temperature 62˚C.
Line 102: Line 112:
pET28a+
pET28a+
-
23/05/2012
+
<h4>23/05/2012</h4>
Repeat what was done on 14/05 (PCR Temp 60˚C + gel extraction.
Repeat what was done on 14/05 (PCR Temp 60˚C + gel extraction.
Line 110: Line 120:
:(
:(
-
16/05/2012
+
<h4>16/05/2012</h4>
PICTUUUURE
PICTUUUURE
Line 122: Line 132:
Still non-specific products observed for the PCR. Possible inter-change during gel extraction; pET28a+ and peGFP got mixed up?
Still non-specific products observed for the PCR. Possible inter-change during gel extraction; pET28a+ and peGFP got mixed up?
-
14/05/2012
+
<h4>14/05/2012</h4>
PCR of pET28a+ and peGFP -> annealing temperature 56˚C. non-specific DNA products obtained for the vector. Increase annealing temperature? Gel extract?
PCR of pET28a+ and peGFP -> annealing temperature 56˚C. non-specific DNA products obtained for the vector. Increase annealing temperature? Gel extract?
Line 138: Line 148:
PCR No. 2 -> pET28a+ and peGFP -> annealing temperature 60˚C.
PCR No. 2 -> pET28a+ and peGFP -> annealing temperature 60˚C.
-
11/05/2012
+
<h4>11/05/2012</h4>
Gel electrophoresis of the PCR reaction performed on 10/05/2012. Non-specific products obtained. Repeat PCR? Change annealing temperature?
Gel electrophoresis of the PCR reaction performed on 10/05/2012. Non-specific products obtained. Repeat PCR? Change annealing temperature?

Revision as of 11:07, 26 September 2012

Contents

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:

  • pSB1AT3+BBa_J45320 (2012 iGEM distribution kit plate 2 Well 5J)
  • BBa_J63009+BBa_J04450 (2012 iGEM distribution kit plate 1 Well 1A)
  • 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"