"

From 2012.igem.org

Revision as of 10:46, 26 October 2012 (view source) Vries189 (Talk | contribs) (→Mutagenesis) ← Older edit		Revision as of 10:48, 26 October 2012 (view source) Vries189 (Talk | contribs) (→Washing column) Newer edit →
Line 4:		Line 4:
	'''QuikChange® Lightning Site-Directed Mutagenesis Kit'''		'''QuikChange® Lightning Site-Directed Mutagenesis Kit'''
-	== Washing column ==	+	== Prepare the sample reaction(s) as indicated below: ==
	<ol>		<ol>
-	<li>Empty waste bin</li>	+	5 μl of 10× reaction buffer
-	<li>Set loop valve on Load Position</li>	+	X μl (10–100 ng) of dsDNA template
-	<li>Set column valve on designated position</li>	+	X μl (125 ng) of oligonucleotide primer #1
-	<li>Set on the pressure alarm: 5mPa</li>	+	X μl (125 ng) of oligonucleotide primer #2
-	<li>Set flow rate on half the maximum the column can handle</li>	+	1 μl of dNTP mix
-	<li>Run the FPLC for about 2 times the column volume</li>	+	1.5 μl of QuikSolution reagent
-	<li>Empty waste bin</li>	+	ddH2O to a final volume of 50 μl
		+	Then add:
		+	1 μl of QuikChange® Lightning Enzyme
	</ol>		</ol>

---

Revision as of 10:48, 26 October 2012

Contents 1 Mutagenesis 1.1 Prepare the sample reaction(s) as indicated below: 1.2 Washing loop 1.3 Loading sample 1.4 Injecting sample onto the column 1.5 Washing column

Mutagenesis

QuikChange® Lightning Site-Directed Mutagenesis Kit

Prepare the sample reaction(s) as indicated below:

5 μl of 10× reaction buffer X μl (10–100 ng) of dsDNA template X μl (125 ng) of oligonucleotide primer #1 X μl (125 ng) of oligonucleotide primer #2 1 μl of dNTP mix 1.5 μl of QuikSolution reagent ddH2O to a final volume of 50 μl Then add: 1 μl of QuikChange® Lightning Enzyme

Washing loop

1. Set flow rate on 0
2. Set loop valve on Inject Position
3. Set column valve on Bypass position
4. Set on the pressure alarm: 5mPa
5. Set flow rate on half the maximum the column can handle
6. Run the FPLC for about 2 times the loop volume

Loading sample

1. Set flow rate on 0
2. Set loop valve on Load Position
3. Inject the sample into the loop with an syringe
4. Keep the syringe in the machine

Injecting sample onto the column

1. Set flow rate on 0
2. Set loop valve on Inject Position
3. Set column valve on designated position
4. Set on the pressure alarm: 5mPa
5. Set flow rate on half the maximum the column can handle
6. Run the FPLC for about 1 times the loop volume
7. Turn on the Frac 900, to collect the samples

Washing column

1. Set flow rate on 0
2. Empty waste bin
3. Set loop valve on Load Position
4. Set column valve on designated position
5. Set on the pressure alarm: 5mPa
6. Set flow rate on half the maximum the column can handle
7. Run the FPLC for about 2 times the column volume
8. Empty waste bin