"

From 2012hs.igem.org

In progress...

Contents 1 Testing of DNA prepared by several methods 2 PCR of promoters and fluorescent protein coding sequences 3 BsaI-digested Miniprep DNA 4 Obtaining the remaining clones

Testing of DNA prepared by several methods

Yeast genomic DNA is commonly prepared using a phenol/chloroform based method. Because both phenol and chloroform are toxic chemicals, we tested several methods for preparing yeast genomic DNA that do not use these chemicals. We compared a method that uses LiOAc to disrupt the yeast cell wall (BioTechniques 50:325-328, 2011) to methods that use the enzyme zymolyase to disrupt the cell wall (a Promega kit or a protocol from Jim Haber’s lab, “JH zym”). The Haber lab protocol produced the greatest yield of genomic DNA, but DNA made using the Promega kit was used in subsequent PCRs because it seemed to produce the most consistent results. 1µl, 2µl, or 5µl of genomic DNA was used in each PCR reaction with LIF1 promoter primers. Following these initial tests, 1µl of yeast genomic DNA was used for subsequent PCRs. The LiOAc method was the shortest and least expensive and we may switch to this method when future DNA preps are needed.

PCR of promoters and fluorescent protein coding sequences

BsaI-digested Miniprep DNA

The samples on these gels contain BsaI-digested miniprep DNA prepared from individual colonies from the transformations above.

Based on the data above, we concluded that the following clones had inserts of the correct size:

Promoters

MET15 yes: B

GAL1 yes: A

GAL1-L yes: A

GAL1-S yes: B

RAD52 yes: B

RAD50 yes: A

RAD54 yes: A, B

RAD55 yes: A, B

MRE11 yes: A, B

XRS2 yes: A, B

REV1 yes: B

TDH3 yes: A, B

LIF1 none

MIG1 yes: B

TUB1 yes: A

CSE4 yes: B

CLN2 yes: B

CLB2 none

ACT1 none

ENO1 none

ENO2 none

TEF2 yes: B

TEF1 none

EAF6 none

HSP12 yes: A, B

HSP26 yes: A

HSC82 none

MSN2 yes: A

MSN4 none

HHO1 yes: A, D

Protein-coding sequences

BFP maybe: A (the insert may be too large)

GFP none

YFP none

mTangerine none

mCherry yes: A (but bottom band seems possibly too intense)

mGrape1 none

Obtaining the remaining clones

Extra colonies were picked from the transformations for the following promoter clones:

LIF1, CLB2, ACT1, ENO1, ENO2, TEF1, EAF6, HSC82, MSN4

DNA from these clones was miniprepped using Qiagen minipreps. The DNA was digested with BsaI as follows:

miniprep DNA: 3µl 10X NEBuffer 4: 2µl 100X BSA: 0.2µl BsaI: 0.5µl H2O: 14.8µl

BsaI digests were incubated at 37°C for 2 hrs and then run on a 2% agarose gel in 1X sodium borate buffer. Return to our main page.