NYMU Taipei/Lab Notes/2007 9 16

From 2007.igem.org

(Difference between revisions)
(Enzyme Digestion)
(Gel separation)
Line 52: Line 52:
**The insert of CinR+HSL 2 is wrong.
**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)
**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)
 +
[[Image:NYMU_Taipei_gel_20070916_plasmid_check_2.JPG|300px]]
 +
*Lane 1: 1kb ladder
 +
*Lane 2, 3: D-term EcoRI, XbaI

Revision as of 14:55, 16 September 2007

Enzyme Digestion

  • 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

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)

NYMU Taipei gel 20070916 plasmid check 2.JPG

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