From 2007.igem.org

Revision as of 07:10, 16 September 2007 (view source) Blent (Talk | contribs) ← Older edit		Revision as of 07:12, 16 September 2007 (view source) Blent (Talk | contribs) Newer edit →
Line 35:		Line 35:
	----		----
	* total gel separation volume is 50 uL		* total gel separation volume is 50 uL
-	** vector weight is 300 ng
	----		----
	* total ligation volume is 10uL		* total ligation volume is 10uL
		+	** vector weight is 300 ng
	** 10X buffer (1uL)		** 10X buffer (1uL)
	** ligase (1uL)		** ligase (1uL)
Line 44:		Line 44:
	* formula		* formula
	** (1) 300 * SI * 3 * 10 (損失因子) / SV = CI * X * L /50		** (1) 300 * SI * 3 * 10 (損失因子) / SV = CI * X * L /50
-	** (2) 300 ng = CV (ng/uL) * VV (uL), VV = 300/CV	+	** (2) 300 ng = CV (ug/uL) * VV (uL) * 1000, VV = 0.3/CV
	** VV (uL) is volume of vector		** VV (uL) is volume of vector
	** CV (ng/uL) is concentration of vector		** CV (ng/uL) is concentration of vector

---

Revision as of 07:12, 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)

---

• total gel separation volume is 50 uL

---

• total ligation volume is 10uL
  • vector weight is 300 ng
  • 10X buffer (1uL)
  • ligase (1uL)
  • VV + IV + water (8uL)

---

• formula
  • (1) 300 * SI * 3 * 10 (損失因子) / SV = CI * X * L /50
  • (2) 300 ng = CV (ug/uL) * VV (uL) * 1000, VV = 0.3/CV
  • VV (uL) is volume of vector
  • CV (ng/uL) is concentration of vector