User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/10/08

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October 8th, 2010
1. Make the following restrictions
 * pSB3K3 + J23101 SpeI-PstI restriction
 * pSB4A5 + MinBP SpeI-PstI restriction


 * SpeI-PstI double restriction methods:
 * DNA -> 5 ul
 * Buffer 2 -> 2 ul (10% of total volume)
 * BSA -> 1 ul
 * SpeI -> 1 ul
 * PstI -> 1 ul
 * HPLC -> 10 ul (to complete total volume of 20ul)
 * Incubate at 37º C for 4 hrs.

2. Make PCR to test the construction:
 * pSB1A2 + J23101 + ΔRBS + GFP E004 (Tubes 1-2)
 * Positive control pSB1C3 (Tube 3)
 * DNA used as template: 2ul of 1:4 dilution of extracted plasmid
 * Primers used were Preffix FWD and Suffix REV, reactives needed for one reactions are as follows:


 * PCR with Taq DNA polymerase
 * Reactive (ul x sample)
 * Taq Polymerase -> 1
 * Taq Reaction Buffer 10X -> 5
 * MgCl 50mM (can be used up to 3ul) -> 2.5
 * dNTP’s 0.4ug/ul -> 2.5
 * Primer Forward (can be used up to 3ul) -> 2.5
 * Primer Reverse (can be used up to 3ul) -> 2.5
 * HPLC -> 32
 * DNA -> 2
 * Total volume -> 50


 * Thermocycler program:
 * 1. 95ºC 5 min
 * 2. 35 cycles
 * -95ºC 45 seg
 * -55ºC 45 seg
 * -72ºC 1:10 min
 * 3. 72ºC 10 min
 * 4. Hold 4ºC

3. Inactivate restriction enzyme for 20 min at 80ºC.

4. Run gel tp verify PCR and restrictions.



Lanes: 1,9) Green ladder; 2) pSB1A2 + J23101 + ΔRBS + GFP E004 (Tube 1); 3) pSB1A2 + J23101 + ΔRBS + GFP E004 (Tube 2); 4) pSB1C3 (Preff FWD-Suff REV); 5) pSB3K3 + J23101 SpeI-PstI restriction; 6) pSB4A5 + MinBP SpeI-PstI restriction; 7) pSB1C3 EcoRI-PstI restriction; 8) ΔRBS + GFP E004 XbaI-PstI restriction.


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