IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/06/21

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DspB Track

Marianne, Vicki

PCR dspB genomic preps

dspB genomic preps arrived from University of Calgary: Blurb from Dave from UofC: 3 strains of A. Pleuropneumoniae sent: H49, H80, and H171. 2 tubes were sent for each strain, for a total of 6 tubes (the duplicates are identical and included for redundancy). The DNA is dissolved in 200 uL of distilled water, and so is PCR-ready, the concentration for each is written on the side, and the samples should be stored at -20 when you receive them. A little more information about the strains: H49 was a veterinary isolate from 1989, H80 is also from '89 and has AP8 in its documentation (may be an official strain designation, I'm not sure though), H171 is from '91 and has the designation 92C (again could be its strain designation, not sure).

The 3 strains of A. Pleuropneumoniae:

H49 x 2. One is 35ng/uL, other is 80ng/uL
AP8 aka H80 x 2. One is 70ng/uL, other is 80ng/uL
92C aka H171 x2. One is 33ng/uL, other 60ng/uL

PCR out dspB using two different primers: one containing His6-tag, and one without the His6-tag.

The following are calculated from the PCR calculator created by last year's iGEM British_Columbia team (2009).

Table 1. Master Mix
REAGENT 1 RXN VOLUME (uL) MASTER MIX (uL): [DNA] in sample
80ng/uL (12 rxns)60ng/uL (6 rxns)
10x rxn buffer2.535.7519.25
10uM FW primer1.2517.8759.625
10uM RE primer1.2517.8759.625
10mM dNTP0.57.153.85
sdH2O18.9875271.52125145.4017
Taq Polymerase0.22.861.54
DNA: 80ng/uL0.31254.46875
DNA: 60ng/uL0.416663.2083
Total: 80ng/uL25357.5
Total: 60ng/uL25192.5

Hot Start PCR protocol can be found here under "Hot Starts"
Making the master mix
The protocol asks to split the reaction into 2 parts.
Part 1:

Table 2. Master Mix B
Master Mix [DNA] in sample; in microcentrifuge tube B
80ng/uL60ng/uL
Polymerase (uL)2.861.54
Buffer (uL)35.7519.25
sdH2O (uL)271.52125145.4017

For each of the respective PCR tubes, pipet 21.6875uL.

Part 2:
Table 3. Master Mix A
Master Mix[DNA] in sample; in microcentrifuge tube A
80ng/uL60ng/uL
RE & FW primer (uL)17.875 x 29.625 x 2
DNA template (uL)4.468723.2083
dNTP (uL)7.153.85

The 60ng/uL Master Mix A is then split into TWO mixes due to one upstream primer having His6-tag and one upstream primer without the His6-tag.

Table 4. Master Mix for 60ng/uL (H171) DNA sample
For 3 rxns For 3 rxns
FW primer + His
4.8125
FW primer
4.8125
RE primer
4.8125
dNTP
1.925
DNA template
1.60415

The 80ng/uL Master Mix A is then split into TWO mixes because there are two different genomic strains: H49 and H80. Then, the master mix for each strain is split into two more mixes (for a total of 4) due to one upstream primer having His6-tag and one upstream primer without the His6-tag.

Table 5. Master Mix for 80ng/uL (H49 and H80) DNA sample
H49H80
RE primer
8.9375
FW primer
8.9375
DNA template
2.234
dNTP
3.575
H49H80
For 3 rxnFor 3 rxnFor 3 rxnFor 3 rxn
FW primer+His
4.46875
FW primer
4.46875
FW primer+His
4.46875
FW primer
4.46875
RE primer
4.46875
DNA template
1.117
dNTP
1.7875

To each PCR tube: pipet 3.3125uL of Master Mix A
Note: For H80 FW primer noHis, may have forgotten to add dNTPVicki Ma 01:26, 28 June 2010 (EDT)

Step 1: Pipet Master Mix A into appropriately labeled PCR tubes

Table 6. PCR tubes - triplets
AH49-His1JH80-1
BH49-His2KH80-2
CH49-His3LH80-3
DH49-1MH171-His-1
EH49-2NH171-His-2
FH49-3OH171-His-3
GH80-His-1PH171-1
HH80-His-2QH171-2
IH80-His-3RH171-3
SdH2O (control)

Step 2: Heat the machine to 95ºC for 2 minutes
Step 3: Pipet Master Mix B respectively into all PCR tubes
Step 4: Resume PCR under the following conditions

Cycle program 30x:
95ºC @ 30s (Denaturing)
65ºC @ 10s (Annealing)
72ºC @ 72s (Extension)
72ºC @ 10 minutes (Final Extension)
10ºC @ Hold (Chill)
  • Start PCR (Hot Start) at 1753
  • Ended PCR (Hot Start) at 1922
  • Store in 4ºC walk-in fridge
    Vicki Ma 01:44, 28 June 2010 (EDT)


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