IGEM:MIT/2007/Notebook/2007-8-16: Difference between revisions
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put into 37C at 12:15 PM | put into 37C at 12:15 PM | ||
For ligation in 1AC3: | |||
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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 |
Revision as of 18:10, 16 August 2007
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