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.

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: