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