NYMU Taipei/Lab Notes/2007 9 16

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< NYMU Taipei/Lab Notes(Difference between revisions)
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==Enzyme Digestion==
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==Enzyme Digestion for CinR+HSL==
*Enzyme digestion: CinR+HSL (with RBS) with EcoRI, SpeI
*Enzyme digestion: CinR+HSL (with RBS) with EcoRI, SpeI
**CinR+HSL (1~4): 14ul
**CinR+HSL (1~4): 14ul
Line 7: Line 7:
**10x EcoRI buffer: 2ul
**10x EcoRI buffer: 2ul
**Total: 20ul
**Total: 20ul
 +
**37° 2h
*Enzyme digestion: D-term with EcoRI
*Enzyme digestion: D-term with EcoRI
** Original we need double digestion EcoRI and XbaI.
** Original we need double digestion EcoRI and XbaI.
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** H2O: 6ul
** H2O: 6ul
** Total: 20ul
** Total: 20ul
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**37°C 2h
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*Enzyme digestion: D-term E with XbaI
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**D-term E: 30ul
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**XbaI: 2ul
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**10x 2 buffer: 5ul
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**10x BSA: 5ul
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**H2O: 8ul
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**Total: 50ul
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**37°C 2h
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==Gel separation==
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==Digestion check of CinR+HSL and D-term by Gel separation==
* 1% gel, TAE 1X, 30 min, 100v
* 1% gel, TAE 1X, 30 min, 100v
** 11 lanes (left most is marker)
** 11 lanes (left most is marker)
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** sample after dye addition is 20 + 4 = 24 uL
** sample after dye addition is 20 + 4 = 24 uL
[[Image:NYMU_Taipei_gel_20070916_plasmid_check_1.JPG|300px]]
[[Image:NYMU_Taipei_gel_20070916_plasmid_check_1.JPG|300px]]
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*Lane 1: 1kb ladder (5ul)
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*Lane 2, 3: CinR+HSL 1 EcoRI, SpeI
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*Lane 4, 5: CinR+HSL 2 EcoRI, SpeI
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*Lane 6, 7: CinR+HSL 3 EcoRI, SpeI
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*Lane 8, 9: CinR+HSL 4 EcoRI, SpeI
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*Lane 10, 11: D-term EcoRI
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*Comments
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**The concentration of CinR+HSL 1 is too low.
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**The insert of CinR+HSL 2 is wrong.
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**EcoRI digestion of D-term is incomplete, hence only the upper band is isolated for DNA extraction. (The lower band is probably the negative supercoiled form of the plasmid)
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== 2nd digestion of D-term and check==
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* D-term EcoRI + XbaI
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[[Image:NYMU_Taipei_gel_20070916_plasmid_check_2.JPG|300px]]
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*Lane 1: 1kb ladder
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*Lane 2, 3: D-term EcoRI, XbaI
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*Lane 4, 5: Empty

Latest revision as of 15:04, 9 October 2007

Enzyme Digestion for CinR+HSL

  • Enzyme digestion: CinR+HSL (with RBS) with EcoRI, SpeI
    • CinR+HSL (1~4): 14ul
      • 因為濃度未知, 所以用最大體積
    • EcoRI: 1ul (20,000 units/ml)
    • SpeI: 1ul (10,000 units/ml)
    • 10x EcoRI buffer: 2ul
    • Total: 20ul
    • 37° 2h
  • Enzyme digestion: D-term with EcoRI
    • Original we need double digestion EcoRI and XbaI.
    • However, NEB recommend to perform the digestion in sequential manner.
    • Thus, we digest the EcoR1 first
    • D-term: 11ul (損失因子10, 目標重量300ng)
      • 300(ng) * 10 = 3(ug) = 0.27 (ug/uL) * X(uL), X = 3/0.27 = 11.11111111
    • EcoRI: 1ul
    • 10x EcoRI buffer: 2ul
    • H2O: 6ul
    • Total: 20ul
    • 37°C 2h
  • Enzyme digestion: D-term E with XbaI
    • D-term E: 30ul
    • XbaI: 2ul
    • 10x 2 buffer: 5ul
    • 10x BSA: 5ul
    • H2O: 8ul
    • Total: 50ul
    • 37°C 2h

Digestion check of CinR+HSL and D-term by Gel separation

  • 1% gel, TAE 1X, 30 min, 100v
    • 11 lanes (left most is marker)
    • each sample (24 uL) are separated into 2 lanes (12 uL for each lane)
      • CinR+HSL #1-#4 (lane 2-9) and D-term (lane 10-11)
  • use 1Kb ladder
    • CinR+HSL insert size = 1.53 Kb
    • D-term vector size = 3.284 Kb
  • 6X dye
    • total volume after digestion is 20uL
    • thus, the dye is 4uL
      • X/(20+X) = 1/6, X = 4
    • sample after dye addition is 20 + 4 = 24 uL

NYMU Taipei gel 20070916 plasmid check 1.JPG

  • Lane 1: 1kb ladder (5ul)
  • Lane 2, 3: CinR+HSL 1 EcoRI, SpeI
  • Lane 4, 5: CinR+HSL 2 EcoRI, SpeI
  • Lane 6, 7: CinR+HSL 3 EcoRI, SpeI
  • Lane 8, 9: CinR+HSL 4 EcoRI, SpeI
  • Lane 10, 11: D-term EcoRI
  • Comments
    • The concentration of CinR+HSL 1 is too low.
    • The insert of CinR+HSL 2 is wrong.
    • EcoRI digestion of D-term is incomplete, hence only the upper band is isolated for DNA extraction. (The lower band is probably the negative supercoiled form of the plasmid)

2nd digestion of D-term and check

  • D-term EcoRI + XbaI

NYMU Taipei gel 20070916 plasmid check 2.JPG

  • Lane 1: 1kb ladder
  • Lane 2, 3: D-term EcoRI, XbaI
  • Lane 4, 5: Empty
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