User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/09/14

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September 14th, 2010
1. Gel to verify PCR made on September 13th.

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Lanes: 1) Green ladder; 2-10) Paz’s samples; 11) BBa_I20260 Suffix FWD-J23101 REV reaction 1; 12) BBa_I20260 Suffix FWD-J23101 REV reaction 2; 13) BBa_I20260 RBS GFP FWD-Suffix REV reaction 1; 14) BBa_I20260 RBS GFP FWD-Suffix REV reaction 2; 15) Green ladder.


 * There are inespecific bands in the gel besides the expected products, I’ll repeat the PCR to try to eliminate these inespecific bands.

2. Repeat PCR to amplify BBa_I20260 (pSB3K3 + J23101 promoter + GFP E0040), one reaction will amplify the plasmid including the J23101 promoter, another reaction will amplify only the GFP E0040 and a third reaction will amplify pSB3K3 plasmid and insert into this plasmid our minimum blue promoter.
 * PCR will be done with Platinum Taq Polymerase.
 * I will make 2 reactions with primers Suffix FWD-J23101 REV (Tubes 1-2).
 * PCR product is expected to be 3670 bp (35 promoter + 2729 plasmid pSB3K3).
 * To amplify the GFP E0040: RBS GFP FWD-Suffix REV (Tubes 3-4) GFP length is 720 bp.
 * PCR to insert minimum blue promoter into pSB3K3 will be done with minBP REV-Suffix FWD primers.
 * Template for the three reactions is BBa_I20260 plasmid diluted 1:4


 * PCR with Platinum Taq DNA polymerase	Volume (ul)
 * 10X PCR Buffer minus M -> 5
 * 10mM dNTP mixture -> 1
 * 50mM MgCl2 -> 1.5
 * Primer mix (10uM each) -> 1
 * Platinum Taq DNA Pol -> 0.4
 * Template DNA -> 2
 * HPLC -> 35.1
 * Total volume -> 50


 * If primers are separated and in concentration 5uM, use 1ul of each one.


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

3. Run gel to verify PCR.



Lanes: 1) Green ladder; 2) BBa_I20260 Suffix FWD-J23101 REV reaction 1 (tube 1); 3) BBa_I20260 Suffix FWD-J23101 REV reaction 2 (tube 2); 4) BBa_I20260 RBS GFP FWD-Suffix REV reaction 1 (tube 3); 5) BBa_I20260 RBS GFP FWD-Suffix REV reaction 2 (tube 4); 6) BBa_I20260 MinBP REV-Suffix FWD primers (tube 5); 7) BBa_I20260 MinBP REV-Suffix FWD primers (tube 6).

4. Purify PCR products from BBa_I20260 Suffix FWD-J23101 REV (reactions 1 and 2) and BBa_I20260 RBS GFP FWD-Suffix REV (reactions 1 and 2) using the High Pure PCR Product Purification Kit Roche.

5. Make restrictions to pure PCR products, plasmid pSB3K3 with promoter J23101 will be restricted with SpeI-PstI and GFP E0040 with XbaI-PstI.
 * SpeI-PstI double restriction methods:
 * DNA -> 15 ul
 * Buffer 2 -> 4 ul (10% of total volume)
 * BSA -> 1 ul
 * SpeI -> 2 ul
 * PstI -> 2 ul
 * HPLC -> 16 ul (to complete total volume of 20ul)


 * XbaI-PstI double restriction methods:
 * DNA -> 15 ul
 * Buffer 2 -> 4 ul (10% of total volume)
 * BSA -> 1 ul
 * XbaI -> 2 ul
 * PstI -> 2 ul
 * HPLC -> 16 ul (to complete total volume of 20ul)


 * Incubate at 37ºC overnight.

6. Repeat PCR to amplify BBa_I20260 (pSB3K3 + J23101 promoter + GFP E0040) and insert into this plasmid our minimum blue promoter.
 * PCR will be done with Platinum Taq Polymerase.
 * I will make 2 reactions with primers Min-BP REV-Suffix FWD (Tubes 1-2).
 * PCR product is expected to be 3670 bp (35 promoter + 2729 plasmid pSB3K3).
 * Template for the reaction is BBa_I20260 plasmid diluted 1:4


 * PCR with Platinum Taq DNA polymerase	Volume (ul)
 * 10X PCR Buffer minus M -> 5
 * 10mM dNTP mixture -> 1
 * 50mM MgCl2 -> 1.5
 * Primer mix (10uM each) -> 1
 * Platinum Taq DNA Pol -> 0.4
 * Template DNA -> 2
 * HPLC -> 35.1
 * Total volume -> 50


 * If primers are separated and in concentration 5uM, use 1ul of each one.


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


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