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	<td>2. &nbsp;&nbsp;&nbsp;&nbsp; 4°C &nbsp;&nbsp;&nbsp;&nbsp; //</td>		<td>2. &nbsp;&nbsp;&nbsp;&nbsp; 4°C &nbsp;&nbsp;&nbsp;&nbsp; //</td>
-	</tr></table><br><br><br><br><br><br><br><br><br>	+	</tr></table><br><br><br><br><br><br><br><br><br><br><br>
-	<div align="justified">Thermocycler Program: a gradient was used between 65°C and 80°C as annealing temperature. This time we used 30 cycles.<div>	+	<div align="justified">Thermocycler Program: a gradient was used between 65°C and 80°C as annealing temperature. This time we used 30 cycles.<br><br>
		+
		+	Afterwards we digested the products of the MutPCR with DpnI for 20min at 36°C and did a heat kill of the enzyme at 80°C for another 20min.
		+	After treating the PCR product with DpnI we did a Transformation into DH10B strain of E.coli using our standard protocol for transformation.

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Revision as of 01:33, 27 September 2012

Labbook - TAL vector

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08/08/12

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1.) MutPCR for pSB1C3 vector backbone in order to remove restriction sites vor BsmBI
2.) Transformation of Sirt2 and HAT5
3.) GGC in order to produce our MammoBrick

1.) MutPCR:

	Tube 1 [µl]	Tube 2 [µl]
Primer MutPCR fo [10 mM]	1	0,25
Primer MutPCR re [10 mM]	1	0,25
dNTPs [10 mM]	1	1
Phusion Buffer HF	1,25	1,25
Phusion Polymerase	0,25	0,25
DMSO	1	1
MgCl2	1	1
Template	1	1
ddH2O	5	6,5

Program for Thermocycler (20 cycles)
1.      95°C    5min
2.      95°C    50s
3.      80°C     50s
4.      72°C     3min
5.      72°C     7min
6.      4°C      //

2.) Transformation of Sirt2 and HAT5 in DH10B starin of E. coli using our standard protocol for transformation. Kanamycin resistance was used

3.) GGC in order to produce our Mammobrick:

Contens	[µl]
BsaI [10 U/µl]	1,5
NEB Buffer 4	1
ATP [10 mM]	1
BSA	1
TALEN fragment [40 ng]	2
exCMV promotor [20 ng]	0,5
PostORF [20 ng]	1,25
PuroORF [20 ng]	0,5
T7 Ligase	0,25
DTT [10 mM]	1
ddH2O	1

Program for Thermocycler (15 cycles)
1.      37°C      5min
2.      20°C      5min

08/09/12

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1.) Gel-Run of MutPCR:

Gel-run without success (no plasmid containing) MutPCR needs to be repeated!!!

2.) Gel-Run of GGC for MammoBrick:

Gel-run without success!!! GGC needs to be repeated!!!

08/10/12

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1.) Repeating GGC in order to produce our MammoBrick:

Contens	[µl]
BsaI [10 U/µl]	1,5
NEB Buffer 4	1
ATP [10 mM]	1
BSA	1
TALEN fragment [40 ng]	2
exCMV promotor [20 ng]	0,5
PostORF [20 ng]	1,25
PuroORF [20 ng]	0,5
T7 Ligase	0,25
DTT [10 mM]	1
ddH2O	1

One reaction was done with DTT as described above and one reaction was done without any DTT and was simply replaced with ddH2O
Thermocycler Program: as described on 08/08/12, but with 20 cycles instead of 15, afterwards heatkill with 80°C for 20min.

08/11/12

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1.) Transformation of MammoBrick vector produced on day before
2.) Transformation of SUV
3.) Repeating MutPCR on pSB1C3 vector backbone

1.) and 2.)
Transformations were done using our standard protocol. DH10B strain of E.coli was used

3.) MutPCR of pSB1C3 vector backbone:
This time the following conditions were used. In comparison to the MutPCR done on 08/08/12 this time no MgCl2 and DMSO was used.

for 20µl	x1 [ul]
Primer MutPCR fow [10 mM]	1
Primer MutPCR rev [10 mM]	1
Phusion Buffer HF	4
Phusion	0,4
Template	0,2
ddH2O	12,3

Program for Thermocycler (15 cycles)
1.      98°C      30s
2.      98°C      10s
2.      65°C-80°C      15s
2.      72°C      45s
2.      72°C      7min
2.      4°C      //