IGEM:MIT/2007/Notebook/2007-8-16

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(DD I14032.B0034 Mixture)
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#DD I14032.B0034 and put it into pSB1AC3 as a blank vector
#DD I14032.B0034 and put it into pSB1AC3 as a blank vector
#Run Polystyrene binding efficiency assay
#Run Polystyrene binding efficiency assay
 +
 +
==Colony Count of last night's transformations==
 +
<pre>
 +
PLATE NAME                    ANTIBIOTIC    # Colonies
 +
F2620.B0034 in 1AC3            Cm                0
 +
                                Kan              0
 +
 +
F2620.B0034 in 1AC3            Cm                0
 +
  (- control, no vector)      Kan              0
 +
 +
F2620.B0034.CPX.B0014 in 1AC3  Cm                400
 +
  cPCR #6, pick #1
 +
 +
F2620.B0034.CPX.B0014 in 1AC3  Cm                ~1050
 +
  cPCR #8, pick #5
 +
 +
I14032.B0034.CPX.B0014 in 1AC3  Cm                0
 +
                                Kan              8
 +
 +
I14032.B0034.CPX.B0014 in 1AC3  Cm                0
 +
  (- control, I.B34 only)      Kan              2
 +
 +
I14032.B0034.CPX.B0014 in 1AC3  Cm                0
 +
  (- control, CPX.B14 only)    Kan              ~50
 +
 +
I10432.B0034.CPX.B0014 in 1AC3  Cm                0
 +
  (- control, no inserts)      Kan              0
 +
</pre>
==Making Electrochemically competent cells==
==Making Electrochemically competent cells==
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==DD I14032.B0034 Mixture==
==DD I14032.B0034 Mixture==
 +
*Need to redo all I14032.B0034 digestions because it was purified using the wrong kit.  Need QIAGEN Nucleotide Removal Kit (insert is 70 long)
 +
For blank (control) vector:
For blank (control) vector:
<br>
<br>

Revision as of 19:54, 16 August 2007

Contents

Agenda

  1. Make electro/chemically competent cells
  2. Transform and plate R0051
  3. Make more DD R0051
  4. Redo R0051.B0034 ligation
  5. DD I14032.B0034 and put it into pSB1AC3 as a blank vector
  6. Run Polystyrene binding efficiency assay

Colony Count of last night's transformations

PLATE NAME                     ANTIBIOTIC    # Colonies
F2620.B0034 in 1AC3             Cm                0
                                Kan               0

F2620.B0034 in 1AC3             Cm                0
   (- control, no vector)       Kan               0

F2620.B0034.CPX.B0014 in 1AC3   Cm                400
   cPCR #6, pick #1

F2620.B0034.CPX.B0014 in 1AC3   Cm                ~1050
   cPCR #8, pick #5

I14032.B0034.CPX.B0014 in 1AC3  Cm                0
                                Kan               8

I14032.B0034.CPX.B0014 in 1AC3  Cm                0
   (- control, I.B34 only)      Kan               2

I14032.B0034.CPX.B0014 in 1AC3  Cm                0
   (- control, CPX.B14 only)    Kan               ~50

I10432.B0034.CPX.B0014 in 1AC3  Cm                0
   (- control, no inserts)      Kan               0

Making Electrochemically competent cells

Using BL-21 (for higher protein expression) Followed TK's OWW protocol.

DD I14032.B0034 Mixture

  • Need to redo all I14032.B0034 digestions because it was purified using the wrong kit. Need QIAGEN Nucleotide Removal Kit (insert is 70 long)

For blank (control) vector:
5 µL I14032.B0034 #4 (225.7 ng/µL)

1 µL EcoRI

1 µL PstI

0.5 µL BSA

5 µL NEB Buffer 3

37.5 µL H20


For 3A assembly with CPX:
5 µL I14032.B0034 #4 (225.7 ng/µL)

1 µL EcoRI

1 µL SpeI

0.5 µL BSA

5 µL NEB Buffer 2

37.5 µL H20

put into 37C at 12:15 PM

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