IGEM:Cambridge/2008/Notebook/Bacillus/2008/08/18

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Current revision (05:57, 28 October 2008) (view source)
(PA with I13522, E0040 and B0034 Preparation)
 
Line 61: Line 61:
* iGEM34 porgramme is used for PCR
* iGEM34 porgramme is used for PCR
Gel:
Gel:
-
** 4μL dye with 20μL PCR product
+
* 4μL dye with 20μL PCR product
-
** 5μL Hyperladder I with 15μL SDW into lane 2
+
* 5μL Hyperladder I with 15μL SDW into lane 2
-
** Lanes 3-7: I13522 Colonies 1-5
+
* Lanes 3-7: I13522 Colonies 1-5
-
** Lane 8: R0040
+
* Lane 8: R0040
-
** Lane 9: Hyperladder I
+
* Lane 9: Hyperladder I
Result:
Result:
* Size of I13522 is not right as the bands are all around 4500bp but it should be 2375bp
* Size of I13522 is not right as the bands are all around 4500bp but it should be 2375bp

Current revision



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Contents

Test Ery efficiency

Since all Ery plates grew, we want to check the antibiotic stock.

- New Ery plate of IA751 (they should die)

- New LB stock (Ery5) with IA751

Single colony plates

- New plates (Cm5 + Amp100) with single colony of ECE188 (from 14/08), ECE189 (from 13/08) and ECE190 (from 14/08)

- Grow the same single colony into LB with antibiotic

Transformation of glycerol stocks of B.S.

The result from the last transformation of glycerol stocks was negative> We tried again with two different protocols.

  • First protocol

- Spin the glycerol stock, throw out the supernatant (just keep about 100μL), add 0.5μL of Medium B

- Incubate about 1h15

- Add 0.6μg of ECE166 (because we managed to transform BS with this vector)

- Incubate 1h30

- Plate on Cm5 plates (DNA less control and transformed cells)

  • Second protocol

- Spin the glycerol stock, throw out the supernatant (just keep about 100μL), add 0.5μL of Medium B

- Add 0.6μg of ECE166 (because we managed to transform BS with this vector)

- Incubate 30min

- Plate on Cm5 plates (transformed cells)


- Plate cells after 10min of incubation (without DNA) on blank plates (to see if they are alive)

PA with I13522, E0040 and B0034 Preparation

  • R0040 TOP10 miniprep from LB culture done by Kevin
  • 5 single colonies of I13522 picked onto Amp100 plate and LB with Amp100 done on Sunday
  • Miniprep of the 5 colonies with sinigle colony PCR
  • PCR followed standard protocol and product is run on 0.8% EtBr E-gel
  • iGEM34 porgramme is used for PCR

Gel:

  • 4μL dye with 20μL PCR product
  • 5μL Hyperladder I with 15μL SDW into lane 2
  • Lanes 3-7: I13522 Colonies 1-5
  • Lane 8: R0040
  • Lane 9: Hyperladder I

Result:

  • Size of I13522 is not right as the bands are all around 4500bp but it should be 2375bp
  • R0040 is of the correct size
  • Transformed chemically compotent TOP10 with B0034 biobrick extract and pUC9 control
  • Plate on Amp100 plates after standard transformation


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