IGEM:MIT/2007/Notebook/2007-8-16
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Agenda
- Make electro/chemically competent cells
- Transform and plate R0051
- Make more DD R0051
- Redo R0051.B0034 ligation
- DD I14032.B0034 and put it into pSB1AC3 as a blank vector
- 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