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Revision as of 14:52, 4 October 2007 (view source) Blent (Talk | contribs) (→ligation setup) ← Older edit		Revision as of 14:54, 4 October 2007 (view source) Blent (Talk | contribs) (→ligation setup) Newer edit →
Line 38:		Line 38:
	** make vector have 100 ng in sample (total volume 20 uL)		** make vector have 100 ng in sample (total volume 20 uL)
	** maximize the insert volume		** maximize the insert volume
-	* ligation I: pCinRHSL (vector) + OmpRBS (insert)	+	<table>
		+	<tr>
		+	<td>ligation I: pCinRHSL (vector) + OmpRBS (insert)</td>
		+	<td>ligation II: pOmpC (vector) + TATA_INSA (insert)</td>
		+	<td>ligation III: CinR+HSL+D-term (vector) + TATB_INSB (insert)</td>
		+	</tr>
		+
		+	<tr><td>
	<table border=1>		<table border=1>
	<tr><td>pCinRHSL (vector)</td><td>3 uL</td></tr>		<tr><td>pCinRHSL (vector)</td><td>3 uL</td></tr>
Line 45:		Line 52:
	<tr><td>T4 ligase</td><td>1 uL</td></tr>		<tr><td>T4 ligase</td><td>1 uL</td></tr>
	</table>		</table>
		+	</td>
-	* ligation II: pOmpC (vector) + TATA_INSA (insert)	+	<td>
	<table border=1>		<table border=1>
	<tr><td>pOmpC (vector)</td><td>4.5 uL</td></tr>		<tr><td>pOmpC (vector)</td><td>4.5 uL</td></tr>
Line 53:		Line 61:
	<tr><td>T4 ligase</td><td>1 uL</td></tr>		<tr><td>T4 ligase</td><td>1 uL</td></tr>
	</table>		</table>
		+	</td>
-	* ligation III: CinR+HSL+D-term (vector) + TATB_INSB (insert)	+	<td>
	<table border=1>		<table border=1>
	<tr><td>CinR+HSL+D-term (vector)</td><td>3 uL</td></tr>		<tr><td>CinR+HSL+D-term (vector)</td><td>3 uL</td></tr>
Line 60:		Line 69:
	<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>
		+	</table>
		+	</td>
		+
		+	</tr>
		+
	</table>		</table>

---

Revision as of 14:54, 4 October 2007

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 100 ng in sample (total volume 20 uL)
  • maximize the insert volume

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) 3 uL OmpRBS (insert) 14 uL 10X buffer 2 uL T4 ligase 1 uL	pOmpC (vector) 4.5 uL TATA_INSA (insert) 12.5 uL 10X buffer 2 uL T4 ligase 1 uL	CinR+HSL+D-term (vector) 3 uL TATB_INSB (insert) 14 uL 10X buffer 2 uL T4 ligase 1 uL