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Revision as of 15:03, 4 October 2007 (view source) Blent (Talk | contribs) (→ligation setup) ← Older edit		Revision as of 15:04, 4 October 2007 (view source) Blent (Talk | contribs) (→ligation setup) Newer edit →
Line 51:		Line 51:
	<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 colspan=2> insert:vector = 1:7</td></tr>	+	<tr><td>insert:vector</td><td>1:7</td></tr>
	</table>		</table>
	</td>		</td>
Line 61:		Line 61:
	<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 colspan=2> insert:vector = 1:5.5</td></tr>	+	<tr><td>insert:vector</td><td>1:5.5</td></tr>
	</table>		</table>
	</td>		</td>
Line 71:		Line 71:
	<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 colspan=2> insert:vector = 1:7</td></tr>	+	<tr><td>insert:vector</td><td>1:7</td></tr>
	</table>		</table>
	</td>		</td>

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Revision as of 15:04, 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 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