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

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

For ligation in 1AC3:
37 µL F2620+B0034 (27.0 ng/µL, blue label says pure 3A pSB3k3 from XL-1)

1 µL EcoRI

1 µL SpeI

0.5 µL BSA

5 µL NEB Buffer 2

5.5 µL H20

put into 37C at 5:40 PM

Nanodrop of Double Digests(BH)

F2620+B0034 (E,P) - 9.6

I14032+B0034 (E,P) - 20.0

I14032+B0034 (E,S) - 15.4

Ligation Mixtures

1µl 1AC3
16.5µl dd F+B (8/16)
2µl 10x T4 ligase buffer
0.5µl ligase
------------------------
20µl Total


1µl 1AC3
9µl dd F+B (8/15)
2µl 10x T4 ligase buffer
0.5µl ligase
7.5µl H2O
------------------------
20µl Total


0µl 1AC3
16.5µl dd F+B (8/16)
2µl 10x T4 ligase buffer
0.5µl ligase
1µl H2O
------------------------
20µl Total


1µl 1AC3
1µl dd I14032+B0034 (E,S)
6µl dd CPX+B0014
2µl 10x T4 ligase buffer
0.5µl ligase
9.5µl H2O
------------------------
20µl Total


1µl 1AC3
1µl dd I14032+B0034 (E,P)
2µl 10x T4 ligase buffer
0.5µl ligase
15.5µl H2O
------------------------
20µl Total


0µl 1AC3
1µl dd I14032+B0034 (E,P)
2µl 10x T4 ligase buffer
0.5µl ligase
16.5µl H2O
------------------------
20µl Total