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Revision as of 23:51, 13 August 2012 (view source) Nili (Talk | contribs) ← Older edit		Revision as of 02:59, 14 August 2012 (view source) Nili (Talk | contribs) Newer edit →
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	'''During the creation.'''<br>		'''During the creation.'''<br>
-	'''Creating parts of Tar methylation region.'''	+	=Creating parts of Tar methylation region.=
	==Date:8/9==		==Date:8/9==
	===Colony PCR===		===Colony PCR===
Line 53:		Line 53:
	#*50cycle		#*50cycle
	#Storage		#Storage
		+
	----		----
		+
		+	==Date:8/13==
		+	===Confirmed of electrophoresis by PCR products and Ligation of the TA vector===
		+	#Electrophoresis
		+	#Check and Colony PCR
		+	#Add to TA vector
		+	#*PCR products 2μL
		+	#*pMD20-Tvector 1μL
		+	#*D2W 2μL
		+	#*Ligation Mighty Mix 5μL
		+	#Cold storage(16℃,30min)
		+	#Storage(-20℃)

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Revision as of 02:59, 14 August 2012

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During the creation.

Contents 1 Creating parts of Tar methylation region. 1.1 Date:8/9 1.1.1 Colony PCR 1.2 Date:8/11 1.2.1 The purified DNA 1.2.2 PCR Production 1.3 Date:8/13 1.3.1 Confirmed of electrophoresis by PCR products and Ligation of the TA vector

Creating parts of Tar methylation region.

Date:8/9

Colony PCR

Reagent

• TaKaRa Ex Taq(5units/μL) 0.5μL
• 10×Ex Taq buffer 10μL
• dNTP Mixture(2.5Meach) 8μL
• Primer F(10μM) 4μL
• Primer R(10μM) 4μL
• Template(E.coli DH5α)

→Reflection:We Took E.coli too many.You should take less E.coli.
Conditions of the thermal cycler

1. 95℃(5min)
2. 94℃(30sec)
3. 61℃(30sec)
4. 71℃(40sec)
5. 72℃(1min)
6. 4℃(Save)
   • 2~4:35cycle→Reflection:The number of cycles was less.So,We increased The number of cycles in 8/11.(50 cycles)

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Date:8/11

The purified DNA

1. Electrophoresis
   • Marker:pigment(buffer) 1μL,DNA molecule 2μL,TE buffer 3μL
   • Sample:pigment(buffer) 1μL,sample 5μL
   • Gel concentration:1.2%,Migration time:30min

→Reflection:Band was less.

1. Storage

PCR Production

1. Electrophoresis
   • The gel check and cut
2. DNA purification
3. Confirmation of electrophoresis
4. PCR
   • 50cycle
5. Storage

---

Date:8/13

Confirmed of electrophoresis by PCR products and Ligation of the TA vector

1. Electrophoresis
2. Check and Colony PCR
3. Add to TA vector
   • PCR products 2μL
   • pMD20-Tvector 1μL
   • D2W 2μL
   • Ligation Mighty Mix 5μL
4. Cold storage(16℃,30min)
5. Storage(-20℃)