Paris/July 21
From 2007.igem.org
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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 | ||
