Thursday, December 04, 2003

Description
The library is a L. esculentum library of VFNT Cherry and Rio Grande-PtoR. The cosmid library was made from EcoRI partially digested total genomic DNA cloned into the BamHI site of pYAC4, which was size fractionated on a CHEF gel and selected for a range of approx. 150-200 Kb. Average insert size of the library ranges from 50 to 400 Kb

Procedure
Test reactions for different primer combinations

Different DNA sources as test templates and later on as controls
- Genomic DNA preparations (e.g. from Mogeor, M-82)
- Cloned DNA fragments after RAPD reaction
- Cloned DNA inserts from a cDNA library after screening.

Using YAC-PCR program and PCR-Mix described under.


YAC Superpool Screening

1 X 96 well microtiter plate, containing DNA preparations from 384 different YAC clones.

PCR Mix
DNA template 1.0 ul
Primer forward 2.0 ul
Primer reverse 2.0 ul
10X PCR buffer 5.0 ul
dNTPs 4.0 ul
Taq DNA polymerase 2 units
H2O 35.0 ul
Total 50.0 ul


YAC-PCR program Step 1 94 C 2.0 min
Step 2 94 C 2.0 min
Step 3 50 C 2.0 min
Step 4 72 C 2.0 min
Step 5 go to step 2 45 cycles
Step 6 72 C 5.0 min
Step 7 4 C endless



Positive YAC Screening
Reactions:


Cracking of yeast cells yeast cells (as template) 5.0 ul
Zymolase (1 mg/ml) 5.0 ul
10 X PCR buffer 2.0 ul
H2O 8.0 ul
Total 20.0 ul
Incubate at 37 C for 30 min


Add PCR mix: Primer forward 1.0 ul
Primer reverse 1.0 ul
10 X PCR buffer 3.0 ul
dNTPs 2.0 ul
Taq DNA Polymerase 1.0 unit
H2O 22.0 ul

Use YAC PCR Program as above.



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