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Revision as of 14:54, 4 October 2007 (view source) Blent (Talk | contribs) (→ligation setup) ← Older edit		Latest revision as of 15:13, 4 October 2007 (view source) Blent (Talk | contribs) (→transformation setup)
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	== ligation setup ==		== ligation setup ==
	* criteria		* criteria
-	** make vector have 100 ng in sample (total volume 20 uL)	+	** make vector have around 100 ng in sample (total volume 20 uL)
-	** maximize the insert volume	+	** maximize the insert volume, and insert is larger than vector
-	<table>	+	<table border=1 align=center>
-	<tr>	+	<tr align=center>
	<td>ligation I: pCinRHSL (vector) + OmpRBS (insert)</td>		<td>ligation I: pCinRHSL (vector) + OmpRBS (insert)</td>
	<td>ligation II: pOmpC (vector) + TATA_INSA (insert)</td>		<td>ligation II: pOmpC (vector) + TATA_INSA (insert)</td>
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	</tr>		</tr>
-	<tr><td>	+	<tr align=center><td>
-	<table border=1>	+	<table border=1 width=100%>
-	<tr><td>pCinRHSL (vector)</td><td>3 uL</td></tr>	+	<tr><td>pCinRHSL (vector, 20 ng/uL)</td><td>3 uL (60 ng)</td></tr>
-	<tr><td>OmpRBS (insert)</td><td>14 uL</td></tr>	+	<tr><td>OmpRBS (insert, 30 ng/uL)</td><td>14 uL (420 ng)</td></tr>
	<tr><td>10X buffer</td><td>2 uL</td></tr>		<tr><td>10X buffer</td><td>2 uL</td></tr>
	<tr><td>T4 ligase</td><td>1 uL</td></tr>		<tr><td>T4 ligase</td><td>1 uL</td></tr>
		+	<tr><td>insert:vector</td><td>1:7</td></tr>
	</table>		</table>
	</td>		</td>
	<td>		<td>
-	<table border=1>	+	<table border=1 width=100%>
-	<tr><td>pOmpC (vector)</td><td>4.5 uL</td></tr>	+	<tr><td>pOmpC (vector, 10 ng/uL)</td><td>4.5 uL (45 ng)</td></tr>
-	<tr><td>TATA_INSA (insert)</td><td>12.5 uL</td></tr>	+	<tr><td>TATA_INSA (insert, 20 ng/uL)</td><td>12.5 uL (250 ng)</td></tr>
	<tr><td>10X buffer</td><td>2 uL</td></tr>		<tr><td>10X buffer</td><td>2 uL</td></tr>
	<tr><td>T4 ligase</td><td>1 uL</td></tr>		<tr><td>T4 ligase</td><td>1 uL</td></tr>
		+	<tr><td>insert:vector</td><td>1:5.5</td></tr>
	</table>		</table>
	</td>		</td>
	<td>		<td>
-	<table border=1>	+	<table border=1 width=100%>
-	<tr><td>CinR+HSL+D-term (vector)</td><td>3 uL</td></tr>	+	<tr><td>CinR+HSL+D-term (vector, 20 ng/uL)</td><td>3 uL (60 ng)</td></tr>
-	<tr><td>TATB_INSB (insert)</td><td>14 uL</td></tr>	+	<tr><td>TATB_INSB (insert, 30 ng/uL)</td><td>14 uL (420 ng)</td></tr>
	<tr><td>10X buffer</td><td>2 uL</td></tr>		<tr><td>10X buffer</td><td>2 uL</td></tr>
	<tr><td>T4 ligase</td><td>1 uL</td></tr>		<tr><td>T4 ligase</td><td>1 uL</td></tr>
		+	<tr><td>insert:vector</td><td>1:7</td></tr>
	</table>		</table>
	</td>		</td>
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	</table>		</table>
		+
		+	== transformation setup ==
		+	* extract 4 uL from ligation mixture (total 20 uL)
		+	* use whole competent cell (total 1 mL) for each ligation
		+	* add ligation mixture into competent cell before it entirely melts and mix well
		+	* 42 C for 45 sec. and put it into iced water (4 C)
		+	* finish within around 5 mins (don't put the competent cell in iced water more than 5 mins)
		+
		+	== Next tasks ==
		+	* check the ligation by PCR
		+	** if the size is correct, perform plasmid extraction
		+	** else, ligate them again

---

Latest revision as of 15:13, 4 October 2007

Contents 1 digestion check of CinR+HSL+D-term,pCinRHSL and pOmpC (vectors) 2 Re-check the concentration of inserts and vectors 3 ligation setup 4 transformation setup 5 Next tasks

digestion check of CinR+HSL+D-term,pCinRHSL and pOmpC (vectors)

lane A: 1kb ladder lane B: CinR+HSL+D-term x 2 lane C: 1kb ladder lane D: pCinRHSL x 2 lane E: pOmpC x 2

• after gel separation, their O.D. was checked. However, it is too low

Re-check the concentration of inserts and vectors

lane A: 1kb ladder lane B: pCinRHSL lane C: pOmpC lane D: CinR+HSL+D-term lane E: 100bp ladder lane F: TATA_INSA lane G: TATB_INSB lane H: OmpRBS

Owing to concentrations of vectors are too low and A260/A280 ratios are not correct (maybe too much ions) thus, we checked the vectors with inserts by PCR to decide how to ligate them

ligation setup

• criteria
  • make vector have around 100 ng in sample (total volume 20 uL)
  • maximize the insert volume, and insert is larger than vector

ligation I: pCinRHSL (vector) + OmpRBS (insert)	ligation II: pOmpC (vector) + TATA_INSA (insert)	ligation III: CinR+HSL+D-term (vector) + TATB_INSB (insert)
pCinRHSL (vector, 20 ng/uL) 3 uL (60 ng) OmpRBS (insert, 30 ng/uL) 14 uL (420 ng) 10X buffer 2 uL T4 ligase 1 uL insert:vector 1:7	pOmpC (vector, 10 ng/uL) 4.5 uL (45 ng) TATA_INSA (insert, 20 ng/uL) 12.5 uL (250 ng) 10X buffer 2 uL T4 ligase 1 uL insert:vector 1:5.5	CinR+HSL+D-term (vector, 20 ng/uL) 3 uL (60 ng) TATB_INSB (insert, 30 ng/uL) 14 uL (420 ng) 10X buffer 2 uL T4 ligase 1 uL insert:vector 1:7

transformation setup

• extract 4 uL from ligation mixture (total 20 uL)
• use whole competent cell (total 1 mL) for each ligation
• add ligation mixture into competent cell before it entirely melts and mix well
• 42 C for 45 sec. and put it into iced water (4 C)
• finish within around 5 mins (don't put the competent cell in iced water more than 5 mins)

Next tasks

• check the ligation by PCR
  • if the size is correct, perform plasmid extraction
  • else, ligate them again