• 1 µl DNA template
• 0,5 µl EcoRI
• 0,5 µl PstI
• 1 µl buffer O

The samples were incubated for 1 h at 37° C.

Gel electrophoresis
	Gel substances 1% Agarose gel 10 µl RedSafe per 50 ml gel 6 µl 2-Log DNA Ladder(0.1-10.0 kb, New England Bioloabs) 6x loading buffer (for samples) Lane   Content     2-6   Control digestion iBB8 K1-5     7   Test-PCR 2xNR HC+LC     iBB8 K1 + 2 chosen for further work.

• 1 µl DNA template
• 1 µl EcoRI-HF
• 1 µl PstI-HF
• 3,6 µl Cut Smart buffer

The samples were incubated for 1 h at 37° C.

Gel electrophoresis
	Gel substances 1% Agarose gel 10 µl RedSafe per 50 ml gel 6 µl 2-Log DNA Ladder(0.1-10.0 kb, New England Bioloabs) 6x loading buffer (for samples) Lane   Content     2+3   pSB1A3 J04450 K1     4   empty     5+6   pSB1A3 J04450 K2     The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 30 µl H2O.

• 5 µl DNA template
• 0,5 µl enzyme 1
• 0,5 µl enzyme 2
• 2 µl Cut Smart buffer
• 12 µl H2O

Samples:

IBB4 K1, iBB6 K1, iBB7 K3 and iBB3 K1 were digested with EcoRI-HF and SpeI-HF.

iBB8K1, iBB4K1, iBB6K1 and iBB1K1 were digested with XbaI and PstI-HF.

The samples were incubated for 1 h at 37° C. Then 1 µl CIP was addend and the samples were incubated for another 1 h at 37° C.

They were purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 30 µl elution buffer.

Liquid cultures and transformation samples from previous day were discarded because of red colonies (Insert J04450). Digestion of BioBricks repeated like 13.5.13, but without CIP.

The samples were purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl elution buffer.

• 1 µl DNA template
• 1 µl EcoRI
• 1 µl PstI
• 2 µl buffer O
• 0,15 µl H2O

Samples:

iBB4 + iBB8

iBB6 + iBB4

iBB7 + iBB6

iBB3 + iBB1

The samples were incubated for 1 h at 37° C.

Gel electrophoresis
	Gel substances 1% Agarose gel 10 µl RedSafe per 50 ml gel 6 µl 2-Log DNA Ladder(0.1-10.0 kb, New England Bioloabs) 6x loading buffer (for samples) Lane   Content   Expectations 2-5   iBB64 K1-4   700 bp 6+7   iBB76 K1-2   1000 bp
	Lane   Content   Expectations 2-3   iBB76 K3-4   1000 bp 4-7   iBB31 K1-4   700 bp
	Lane   Content   Expectations 2-5   iBB48 K1-4   1900 bp

• 10 µl DNA template
• 0,5 µl enzyme 1
• 0,5 µl enzyme 2
• 2 µl Cut Smart buffer
• 7 µl H2O

Samples:

Digest of pSB1A3 iBB6+4 K1+2 and pSB1A3 iBB3+1 K1+4 with XbaI and PstI-HF

PSB1A3 iBB4+8 K1+4 and pSB1A3 iBB7+6 K2+3 with EcoRI-HF and SpeI-HF.

The samples were incubated for 1 h at 37° C.

They were purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl H2O.

• 1 µl DNA template
• 0,5 µl EcoRI-HF
• 0,5 µl PstI-HF
• 1 µl Cut Smart buffer
• 7 µl H2O

The samples were incubated for 1 h at 37° C.

Gel electrophoresis
	Gel substances 1% Agarose gel 10 µl RedSafe per 50 ml gel 6 µl 2-Log DNA Ladder(0.1-10.0 kb, New England Bioloabs) 6x loading buffer (for samples) Lane   Content   Expectations 2-5   iBB7631 K1-4   Vector: 2150 bp Insert: 1610 bp

• 5 µl DNA template
• 0,5 µl enzyme 1
• 0,5 µl enzyme 2
• 2 µl Cut Smart buffer
• 12 µl H2O

Samples:

Digestion of pSB1C3 iBB6, iBB3, iBB5 and iBB4 with XbaI and PstI-HF

Digestion of pSB1C3 iBB6 and iBB1 with EcoRI-HF and SpeI-HF.

The samples were incubated for 1 h at 37° C.

They were purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl H2O.

• 10 µl DNA template (pSB1C3 J04450)
• 0,5 µl EcoRI-HF
• 0,5 µl PstI-HF
• 2 µl Cut Smart buffer
• 6 µl H2O

The samples were incubated oN at 37° C.

Gel electrophoresis (done 28.05.2013)
	Gel substances 1% Agarose gel 10 µl RedSafe per 50 ml gel 6 µl 2-Log DNA Ladder(0.1-10.0 kb, New England Bioloabs) 6x loading buffer (for samples) Lane   Content     2   pSB1C3 J04450 (EcoRI/PstI cut)     The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 30 µl H2O.

• 1 µl DNA templates (see above)
• 0,5 µl EcoRI
• 0,5 µl PstI-HF
• 1 µl Cut Smart buffer
• 7 µl H2O

The samples were incubated for 1 h at 37° C.

Gel electrophoresis
	Gel substances 1% Agarose gel 10 µl RedSafe per 50 ml gel 6 µl 2-Log DNA Ladder(0.1-10.0 kb, New England Bioloabs) 6x loading buffer (for samples) Lane   Content   Expectations 2+3   iBB63 K1-2   500 bp 4+5   iBB15 K1-2   700 bp
	Lane   Content   Expectations 2+3   iBB54 K1-2   750 bp 4+5   iBB16 K1-2   650 bp

• 29,5 µl DNA templates (PCR-products)
• 1 µl EcoRI-HF
• 1 µl PstI-HF
• 3,5 µl Cut Smart buffer
• 0,5 µl H2O

The samples were incubated for 1 h at 37° C.

They were purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl H2O.