Team:Freiburg/Notebook/26 July

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==<span style="color:blue;">blue light receptor</span>==

===Gibson-Assembly===

'''Investigators: Sandra, Sophie'''

Planned Gibson-Assembly for Lov-tap and Not-Gate. Need to order NAD. We will start an thursday.



==<span style="color:grey;">Precipitator</span>==

'''Ligation'''


{| style="border-spacing:0;"
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name: Ruediger
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date: 26.07

|-
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Continue from Date Name 

Experiment Digestion 25.07 Ruediger

|-
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name:GFPPbd

|}
'''Procedure'''


PCR tube:

total volume 20 μl


# add H<sub>2</sub>O (17 μl -X-Y-Z)
# add 2 μl Ligase Buffer 10x 
# add Insert 1, Insert 2(when proceeding from 3A digestion use 2 μl of each)
# add Vector (20ng needed. When proceeding from 3A digestion use 2 μl)
# Add 1 μl T4-DNA Ligase 
# Incubate 10-30 min at room temperature
# heat for 20 minutes at 80°C
# store at -20°C or directly proceed to transformation


{| style="border-spacing:0;"
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''Part name'''
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''Volume ('''μl)
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''Part name'''
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''Volume ('''μl)

|-
| style="border-top:0.0069in solid #00000a;border-bottom:none;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''S39'''
| style="border-top:0.0069in solid #00000a;border-bottom:none;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''2'''
| style="border-top:0.0069in solid #00000a;border-bottom:none;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''S43'''
| style="border-top:0.0069in solid #00000a;border-bottom:none;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''2'''

|-
| style="border-top:none;border-bottom:none;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''P18'''
| style="border-top:none;border-bottom:none;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''1,8'''
| style="border-top:none;border-bottom:none;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''P18'''
| style="border-top:none;border-bottom:none;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''1,8'''

|-
| style="border-top:none;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''Psb1T3'''
| style="border-top:none;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''2'''
| style="border-top:none;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''Psb1T3'''
| style="border-top:none;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''2'''

|-
| style="border-top:0.0069in solid #00000a;border-bottom:none;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''S39'''
| style="border-top:0.0069in solid #00000a;border-bottom:none;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''2'''
| style="border-top:0.0069in solid #00000a;border-bottom:none;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''S43'''
| style="border-top:0.0069in solid #00000a;border-bottom:none;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''2'''

|-
| style="border-top:none;border-bottom:none;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''P19'''
| style="border-top:none;border-bottom:none;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''1,8'''
| style="border-top:none;border-bottom:none;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''P19'''
| style="border-top:none;border-bottom:none;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''1,8'''

|-
| style="border-top:none;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''Psb1T3'''
| style="border-top:none;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''2'''
| style="border-top:none;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''Psb1T3'''
| style="border-top:none;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''2'''

|-
| style="border-top:0.0069in solid #00000a;border-bottom:none;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''S39'''
| style="border-top:0.0069in solid #00000a;border-bottom:none;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''2'''
| style="border-top:0.0069in solid #00000a;border-bottom:none;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''S43'''
| style="border-top:0.0069in solid #00000a;border-bottom:none;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''2'''

|-
| style="border-top:none;border-bottom:none;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''P20'''
| style="border-top:none;border-bottom:none;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''1,8'''
| style="border-top:none;border-bottom:none;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''P20'''
| style="border-top:none;border-bottom:none;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''1,8'''

|-
| style="border-top:none;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''Psb1T3'''
| style="border-top:none;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''2'''
| style="border-top:none;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''Psb1T3'''
| style="border-top:none;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''2'''

|}
'''Added 12,2''' μl to every sample


'''Documentation:'''

Why are you doing this experiment? Where are your parts stored? Name the parts for ligation etc.


{| style="border-spacing:0;"
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| See digestion yesterday

3A assembly combination as shown in table.

|}


Transformation''' '''


{| style="border-spacing:0;"
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name: RT
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date: 26.07

|-
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Continue from Date Name 

Experiment Ligation 26.07 Ruediger

|-
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name: GFPPbd

|}
Procedure


# take cells from -80°C freezer and put them on ice! (every eppi contains about 400 μl cells)
# thaw cells on ice 20 minutes
# pipette 50 μl cells and 2 μl DNA into eppi still on ice!
# Incubate for 30 minutes on ice
# Heat at 42°C for 60 sec
# Incubate on ice for 5 minutes
# Add 200 μl LB Broth
# Incubate for 2 hours at 37°C (cells with lysis cassette at 30°C!!)
# Plate 50 μl and 200μl on two different LB/Agar plates with appropriate antibiotic resistance

'''Documentation:'''

Why are you doing this experiment? Name of the sample? Where are they stored? Name the vector with inserts, antibiotika resistance etc.


{| style="border-spacing:0;"
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Had only 6 tet plates -> put 100 μl of each sample on each plate

Incubator overnight for 30°C


S39-P18

S39-P19

S39-P20


S43-P18

S43-P19

S43-P20

|}

<br/>

==<span style="color:orange;">Lysis cassette</span>==

===Sequencing of Quickchange-modified Lysis Repressor Part (modified K098995)===

[https://static.igem.org/mediawiki/2011/3/36/Freiburg11_seq_qcLysepromotor_2607.gb Right-click to download the annotated ape (.gb) file]

<br>

===3A Assembly (K098995 + K124017)===

'''Digestion of 3A Assembly'''


{| style="border-spacing:0;"
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name: Theo
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date: 26.07.2011

|-
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Continue from Experiment : Quickchange V.3 

|-
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name: Lysis Cassette V.2 (3A assembly of quickchange-modified K098995 with Lysis genes K124017)

|}
Procedure


# add H<sub>2</sub>O (38μl-DNA ) 
# 5 μl NEB4 buffer  (stored at iGEM’s, -20°C)
# 5 μl 10x BSA  (used 1:10 diluted sample stored at iGEM’s, -20°C)
# DNA (500 ng) (Vector:Insert ratio 1:3 in following Ligation)
# 1 μl restriction enzymes  (stored at iGEM’s, -20°C)
# heat for 1-2 hours 37°C (6 hours if time)
# heat for 20 minutes 80°C (inactivation of enzymes)
# keep at 4°C if you cannot continue

Restriction enzymes you need to cut the vector, insert1 and insert 2:


{| style="border-spacing:0;"
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''Components'''
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>'''Vector (μl)'''</center>
| colspan="3"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>'''Insert1 -'''K098995-''' and 2 -''' K124017'''-(μl)'''</center>

|-
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| DNA (500ng)
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 10
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 5
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 7

|-
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| BSA (10x) (5μl)
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 

|-
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| NEB4 Buffer (5μl)
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 

|-
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Enzyme 1 (1μl)
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| EcoRI + DpnI
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| EcoRI
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| XbaI

|-
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Enzyme 2 (1μl)
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| PstI
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| SpeI
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| PstI

|-
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| H<sub>2</sub>O (38 μl- DNA)
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 27
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 33
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 31

|-
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| In total 50 μl
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 

|}
'''Documentation:'''

Why are you doing this experiment? Where are the samples stored? Antibiotica resistance, vector used etc.


{| style="border-spacing:0;"
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 3A assembly of quickchange-modified K098995 with Lysis genes K124017.

Quickchange-modified K098995: 132,8ng/µl (Amp)

Lysis genes K124017: (Amp+Kan)

<nowiki>*Parts were run on the gel and verified that the inserts were cut, image accidentaly</nowiki> not saved* 

Vector: pSB1T3 + DpnI Digestion

Stored in Theos Box

|}

<br>
<br/>

'''Ligation'''


{| style="border-spacing:0;"
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name: Theo
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date: 26.07.2011

|-
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Continue from: 26.07.2011 Lysis Cassette V.2 Digestion 

|-
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name: Lysis Cassette V.2 (3A assembly of quickchange-modified K098995 with Lysis genes K124017)

|}
'''Procedure'''


PCR tube:

total volume 20 μl


# add H<sub>2</sub>O (17 μl -X-Y-Z)
# add 2 μl Ligase Buffer 10x 
# add Insert 1, Insert 2(when proceeding from 3A digestion use 2 μl of each)
# add Vector (20ng needed. When proceeding from 3A digestion use 2 μl)
# Add 1 μl T4-DNA Ligase 
# Incubate 10-30 min at room temperature
# heat for 20 minutes at 80°C
# store at -20°C or directly proceed to transformation


{| style="border-spacing:0;"
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name of part
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Ratio Insert:Vector

<nowiki>= 3:1 or 1:1</nowiki>
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Volume (μl)

|-
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| X insert 1
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Modif. K098995
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 3:1 (1,5*3)
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 4,5

|-
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Y insert 2
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| K124017
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 3:1 (2*3)
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 6

|-
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Z vector
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| pSB1T3
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1:3 (1)
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1

|-
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| H<sub>2</sub>O
| style="background-color:#bfbfbf;border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 
| style="background-color:#bfbfbf;border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 5,5

|}
'''Documentation:'''

Why are you doing this experiment? Where are your parts stored? Name the parts for ligation etc.


{| style="border-spacing:0;"
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Ligation step of 3A assembly.


How to calculate ratios --> e.g. for K124017

Length of pSB1T3= ca 2200bp

Length of K124017+Vector<nowiki>= ca 4500bp</nowiki>

4500/2200= ca 2

So 2*3 µl (since 3:1 is needed) = 6 µl




|}

<br>
<br/>

'''Transformation'''


{| style="border-spacing:0;"
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name: Theo
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date: 

|-
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Continue from : 26.07.2011 Lysis Cassette V.2 Ligation 




|-
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name: Lysis Cassette V.2 (3A assembly of quickchange-modified K098995 with Lysis genes K124017)

|}
Procedure


# take cells from -80°C freezer and put them on ice! (every eppi contains about 400 μl cells)
# thaw cells on ice 20 minutes
# pipette 50 μl cells and 2 μl DNA into eppi still on ice!
# Incubate for 30 minutes on ice
# Heat at 42°C for 60 sec
# Incubate on ice for 5 minutes
# Add 200 μl LB Broth
# Incubate for 2 hours at 37°C (cells with lysis cassette at 30°C!!)
# Plate 50 μl and 200μl on two different LB/Agar plates with appropriate antibiotic resistance

'''Documentation:'''

Why are you doing this experiment? Name of the sample? Where are they stored? Name the vector with inserts, antibiotika resistance etc.


{| style="border-spacing:0;"
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| New Composite Part in pSB1T3 Vector with quickchange-modified K098995 and Lysis genes K124017

|}

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