Paris/July 21

From 2007.igem.org

< Paris(Difference between revisions)
 
(13 intermediate revisions not shown)
Line 1: Line 1:
 +
[[Paris/July 20|yesterday]] -- [[Paris/July 22|tomorrow]] <br>
== Digestion reactions ==
== Digestion reactions ==
 +
 +
Double digestion reactions were performed as follows:
 +
for a double digestion by X & Y, the template was digested by X(mix1:in a 25µL reaction mix)& Y (mix2: in a 25µL reaction mix) for 2h at 37°. Then, enzyme Y was added to mix1 & enz. X added to mix 2. This second reaction was also performed at 37° for 2h.
 +
 +
 +
25µL reaction mix:
 +
*10µL DNA template (miniprep or PCR product)
 +
*1µL enz. (added last)
 +
*2.5µL 10x reaction buffer (depending on enzymes used)
 +
*BSA 100x 0.25µL
 +
*H2O qsp 25µL
 +
 +
{|
{|
|- style="background: #ccccff;"
|- style="background: #ccccff;"
Line 9: Line 23:
|width=5%| Enzyme 1
|width=5%| Enzyme 1
|width=5%| Enzyme 2
|width=5%| Enzyme 2
-
|width=40%| Description
+
|width=5%| size
 +
|width=35%| Description
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
| style="background: #ccffcc;" |D9
| style="background: #ccffcc;" |D9
Line 16: Line 31:
|EcoI
|EcoI
|SpeI
|SpeI
-
|Vector with the J23100 promoter ready for insertion of a RBS/CDS backward-insert  
+
|2096
 +
|Vector with mRFP RBS/CDS ready for insertion promoter (fw-insert)
 +
|
 +
|
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
| style="background: #ccffcc;" |D10
| style="background: #ccffcc;" |D10
Line 23: Line 41:
|EcoI
|EcoI
|SpeI
|SpeI
-
|J23100 promoter ready for usage as a fw-insert
+
|58
 +
|J23100 promoter ready for use as a fw-insert
 +
|
 +
|
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
| style="background: #ccffcc;" |D11
| style="background: #ccffcc;" |D11
Line 30: Line 51:
|EcoRI
|EcoRI
|SpeI
|SpeI
-
|Vector with the J23107 promoter ready for insertion of a RBS/CDS backward-inser
+
|2096
 +
|Vector with mRFP RBS/CDS ready for insertion of a fw-insert promoter
 +
|
 +
|
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
| style="background: #ccffcc;" |D12
| style="background: #ccffcc;" |D12
|<bbpart>pJ23107</bbpart> promoter fw-insert
|<bbpart>pJ23107</bbpart> promoter fw-insert
-
|<bbpart>pJ23100</bbpart> (MP2.1, MP2.2, MP2.3 & MP2.4)
+
|<bbpart>pJ23107</bbpart> (MP2.1, MP2.2, MP2.3 & MP2.4)
|EcoRI
|EcoRI
|SpeI
|SpeI
 +
|58
|J23107 promoter ready for usage as a fw-insert
|J23107 promoter ready for usage as a fw-insert
 +
|
 +
|
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
| style="background: #ccffcc;" |D13
| style="background: #ccffcc;" |D13
|<bbpart>mRFP</bbpart>  
|<bbpart>mRFP</bbpart>  
-
|<bbpart>pJ23100</bbpart> MP2.2
+
|<bbpart>pJ23107</bbpart> (MP2.2, MP2.3 & MP2.4)
|SpeI
|SpeI
|PstI
|PstI
-
|mRFP RBS/CDS/T sequence, can it be used as a backward inser?
+
|887
 +
|mRFP RBS/CDS/T sequence, can it be used as a backward insert? => yes but you will not be able to add any biobrick after it
 +
|
 +
|
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
| style="background: #ccffcc;" |D14
| style="background: #ccffcc;" |D14
Line 51: Line 81:
|XbaI
|XbaI
|PstI
|PstI
 +
|1063
|Cre ORF extracted for usage as a bw-insert
|Cre ORF extracted for usage as a bw-insert
 +
|
 +
|
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
-
| style="background: #ccffcc;" |D14
+
| style="background: #ccffcc;" |D15
|<bbpart>B0030</bbpart> bw-vector
|<bbpart>B0030</bbpart> bw-vector
|<bbpart>B0030</bbpart> MP5.1 & MP5.2
|<bbpart>B0030</bbpart> MP5.1 & MP5.2
|SpeI
|SpeI
|PstI
|PstI
 +
|2076
|bacward vector for cloning a CDS bw-insert after the B0030 RBS
|bacward vector for cloning a CDS bw-insert after the B0030 RBS
 +
|
 +
|
|- style="background: #cccccc;"   
|- style="background: #cccccc;"   
-
| style="background: #ccffcc;" |D15
+
| style="background: #ccffcc;" |D16
|<bbpart>pSB1AK3</bbpart> vector
|<bbpart>pSB1AK3</bbpart> vector
|<bbpart>B0015</bbpart> MP3.1 & MP3.2
|<bbpart>B0015</bbpart> MP3.1 & MP3.2
|EcoRI
|EcoRI
|PstI
|PstI
 +
|3148
|pSB1AK3 open vector for BioBrick cloning
|pSB1AK3 open vector for BioBrick cloning
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D17
 +
|pSB1A2 <bbpart>E0422</bbpart> vector
 +
|<bbpart>E0422</bbpart> (MP6.1 & MP6.2)
 +
|EcoRI
 +
|PstI
 +
|2038
 +
|pSB1A2 open vector for BioBrick cloning
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D18
 +
|BB dig. lox66DapAE.coli
 +
|lox66-DapAE.coli P5 PCR product
 +
|EcoRI
 +
|PstI
 +
|~960
 +
|PCR product digested for cloning as a BioBrick
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D19
 +
|BB dig. lox66DapAsubtilis
 +
|lox66-DapAsubtilis P6 PCR product
 +
|EcoRI
 +
|PstI
 +
|~960
 +
|PCR product digested for cloning as a BioBrick
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D20
 +
|fw insert lox66-DapAE.coli
 +
|lox66-DapAE.coli P5 PCR product
 +
|XbaI
 +
|PstI
 +
|~960
 +
|PCR product digested for usage as a forward insert
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D21
 +
|fw insert lox66-DapAsubtilis
 +
|lox66-DapAsubtilis P6 PCR product
 +
|XbaI
 +
|PstI
 +
|~960
 +
|PCR product digested for usage as a forward insert
 +
|
 +
|
|}
|}

Latest revision as of 17:49, 7 October 2007

yesterday -- tomorrow

Digestion reactions

Double digestion reactions were performed as follows: for a double digestion by X & Y, the template was digested by X(mix1:in a 25µL reaction mix)& Y (mix2: in a 25µL reaction mix) for 2h at 37°. Then, enzyme Y was added to mix1 & enz. X added to mix 2. This second reaction was also performed at 37° for 2h.


25µL reaction mix:

  • 10µL DNA template (miniprep or PCR product)
  • 1µL enz. (added last)
  • 2.5µL 10x reaction buffer (depending on enzymes used)
  • BSA 100x 0.25µL
  • H2O qsp 25µL


Digestion Products
Number Product Name Matrix Name Enzyme 1 Enzyme 2 size Description
D9 pJ23100 ES vector pJ23100 (MP1.1 & MP1.2) EcoI SpeI 2096 Vector with mRFP RBS/CDS ready for insertion promoter (fw-insert)
D10 pJ23100 promoter fw-insert pJ23100 (MP1.1 & MP1.2) EcoI SpeI 58 J23100 promoter ready for use as a fw-insert
D11 pJ23107 ES vector pJ23107 (MP2.1, MP2.2, MP2.3 & MP2.4) EcoRI SpeI 2096 Vector with mRFP RBS/CDS ready for insertion of a fw-insert promoter
D12 pJ23107 promoter fw-insert pJ23107 (MP2.1, MP2.2, MP2.3 & MP2.4) EcoRI SpeI 58 J23107 promoter ready for usage as a fw-insert
D13 mRFP pJ23107 (MP2.2, MP2.3 & MP2.4) SpeI PstI 887 mRFP RBS/CDS/T sequence, can it be used as a backward insert? => yes but you will not be able to add any biobrick after it
D14 Cre ORF J61047 MP9.1 & MP9.2 XbaI PstI 1063 Cre ORF extracted for usage as a bw-insert
D15 B0030 bw-vector B0030 MP5.1 & MP5.2 SpeI PstI 2076 bacward vector for cloning a CDS bw-insert after the B0030 RBS
D16 pSB1AK3 vector B0015 MP3.1 & MP3.2 EcoRI PstI 3148 pSB1AK3 open vector for BioBrick cloning
D17 pSB1A2 E0422 vector E0422 (MP6.1 & MP6.2) EcoRI PstI 2038 pSB1A2 open vector for BioBrick cloning
D18 BB dig. lox66DapAE.coli lox66-DapAE.coli P5 PCR product EcoRI PstI ~960 PCR product digested for cloning as a BioBrick
D19 BB dig. lox66DapAsubtilis lox66-DapAsubtilis P6 PCR product EcoRI PstI ~960 PCR product digested for cloning as a BioBrick
D20 fw insert lox66-DapAE.coli lox66-DapAE.coli P5 PCR product XbaI PstI ~960 PCR product digested for usage as a forward insert
D21 fw insert lox66-DapAsubtilis lox66-DapAsubtilis P6 PCR product XbaI PstI ~960 PCR product digested for usage as a forward insert
Retrieved from "http://2007.igem.org/wiki/index.php/Paris/July_21"