NYMU Taipei/phptest

From 2007.igem.org

(Difference between revisions)
Line 33: Line 33:
** BSA (2uL)
** BSA (2uL)
** plasmid + ddH2O (14uL = VI + water, 1 <= VI <= 14)
** plasmid + ddH2O (14uL = VI + water, 1 <= VI <= 14)
-
** 300 ng = CV (ug/uL) * VV (uL) * 1000, VV = 0.3/CV
+
** for vector
 +
*** we assume 300 ng for digestion and 90% loss
 +
*** thus, amplify 10X from 300 ng to 3,000 ng = 3 mg
 +
*** 3,000 ng = CV (ug/uL) * 1000 (ng/ug) * DVV, DVV (digested vector volume) = 3/CV
----
----
* total gel separation volume is 50 uL
* total gel separation volume is 50 uL

Revision as of 09:47, 16 September 2007

size (bp) of insert part (SI)
size (bp) of vector part (SV)
concentration (ug/uL) of insert (CI)
concentration (ug/uL) of vector (CV)
volume (uL) of insert for gel separation (VI)
volume (uL) of insert for ligation (L)

  • total digestion volume is 20 uL (typical)
    • enzyme 1 and enzyme 2 (1uL * 2)
    • 10X buffer (2uL)
    • BSA (2uL)
    • plasmid + ddH2O (14uL = VI + water, 1 <= VI <= 14)
    • for vector
      • we assume 300 ng for digestion and 90% loss
      • thus, amplify 10X from 300 ng to 3,000 ng = 3 mg
      • 3,000 ng = CV (ug/uL) * 1000 (ng/ug) * DVV, DVV (digested vector volume) = 3/CV
  • total gel separation volume is 50 uL
  • total ligation volume is 10uL
    • vector weight is 300 ng
    • 10X buffer (1uL)
    • ligase (1uL)
    • VV + L + water (8uL)
  • formula
    • 300 * SI * 3 * 10 (損失因子) / SV = CI * VI * L /50
    • vector weight is 300 ng
    • SI (bp) is size of insert
    • SV (bp) is size of vector
    • VI (uL) is volume of insert
    • VV (uL) is volume of vector
    • CI (ng/uL) is concentration of insert
    • CV (ng/uL) is concentration of vector
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