생유기화학실험 12주차
Protein Expression
E.Coli transformation
& Purification
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Lac operon
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IPTG induction
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T7 RNA Polymerase
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Materials & Methods – Cell growth
-
LB Broth (pH 7.0)
1000X Ampicillin
BL21-pAR1219
1000X IPTG
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Materials & Methods – Cell lysis
-
Buffer LB
Egg white lysozyme (in buffer LB)
PMSF
Leupeptin
Sodium deoxycholate
Ammonium sulfate
Polyethylene Imine (PEI)
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Materials & Methods – Cell lysis
1. Thaw cells on ice and suspend in 70 mL
cold Buffer LB
2. Add 17 mL of fresh egg white lysozyme, 150
uL PMSF, and 70 uL leupeptin
3. Incubate 20 min on ice
4. Add 9 mL of 0.8% sodium deoxycholate and
incubate for another 20 min
5. Add 150 uL PMSF, 17 mL 2M Ammonium
sulfate, add buffer LB up to 170 mL.
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Materials & Methods – Cell lysis
1. Add 17 mL of 10% PEI slowly on ice and incubate on
ice for 20 min.
2. Centrifuge the lysate to remove cell debris.
3. Collect the supernatant in fresh tube.
4. Slowly add 0.82 volume of saturated ammonium
sulfate (pH 7, 4 oC). DO NOT ADD QUICKLY!!
5. Stir for 20 min on ice
6. Centrifuge and remove supernatant.
7. Dissolve the precipitate in 50 mL of buffer C + 100mM
NaCl
8. Dialyze the sample in 4 L Buffer C + 100 mM NaCl
O/N. Add 4 mL PMSF to dialysis buffer.
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Result and Discussion
- 이번주는 결과 레포트가 없습니다.
- 대신 Materials & method, 간단한 discussion은
노트에 적어서 같이 내길 바랍니다.
- Discussion 에 들 어 갈 내 용 은 , 실 험 하 면 서
자기가 궁금했던 점이라든지, 잘못한 부분,
추가로 조사한 내용 등을 적으면 됩니다.
- 레포트 제출은 금요일 오후 6시까지 산학
717호로 제출하세요.
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Next week
Protein purification (Column chromatography)
- Column chromatography란? (원리, 종류)
- 그 중에서도 ion exchange column을 중심으로
조사할 것
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