Paris/July 21
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| + | [[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 58: | Line 72: | ||
|PstI | |PstI | ||
|887 | |887 | ||
| - | |mRFP RBS/CDS/T sequence, can it be used as a backward insert? => yes but you will not be able to any biobrick after it | + | |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 |
| | | | ||
| | | | ||
| Line 87: | Line 101: | ||
|EcoRI | |EcoRI | ||
|PstI | |PstI | ||
| - | | | + | |3148 |
|pSB1AK3 open vector for BioBrick cloning | |pSB1AK3 open vector for BioBrick cloning | ||
| | | | ||
| Line 97: | Line 111: | ||
|EcoRI | |EcoRI | ||
|PstI | |PstI | ||
| - | | | + | |2038 |
|pSB1A2 open vector for BioBrick cloning | |pSB1A2 open vector for BioBrick cloning | ||
| | | | ||
| Line 107: | Line 121: | ||
|EcoRI | |EcoRI | ||
|PstI | |PstI | ||
| - | | | + | |~960 |
|PCR product digested for cloning as a BioBrick | |PCR product digested for cloning as a BioBrick | ||
| | | | ||
| Line 117: | Line 131: | ||
|EcoRI | |EcoRI | ||
|PstI | |PstI | ||
| - | | | + | |~960 |
|PCR product digested for cloning as a BioBrick | |PCR product digested for cloning as a BioBrick | ||
| | | | ||
| Line 127: | Line 141: | ||
|XbaI | |XbaI | ||
|PstI | |PstI | ||
| - | | | + | |~960 |
|PCR product digested for usage as a forward insert | |PCR product digested for usage as a forward insert | ||
| | | | ||
| Line 137: | Line 151: | ||
|XbaI | |XbaI | ||
|PstI | |PstI | ||
| - | | | + | |~960 |
|PCR product digested for usage as a forward insert | |PCR product digested for usage as a forward insert | ||
| | | | ||
| | | | ||
|} | |} | ||
Latest revision as of 17:49, 7 October 2007
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 | ||
