Gel-stab PCR was carried out to obtain a particular PCR product from a sample which produced more than one product. A band resulting from electrophoresis (corresponding to a particular PCR product) was pierced with a pipette tip (20-200 IlL), and the tip then placed into a microcentrifuge tube containing 200 j..!L of sterile dHzO. The solution was mixed thoroughly, and a S ilL aliquot used per 25 IlL PCR reaction (section 2.8. 1).
Chapter 2 Table 2.2 PCR and Sequencing Primers
Primer Size Tm Sequence (5' to 3') Source
CPD1 24 77 AGGCCTCGAGGCATCAAGGCACr� This
CPD2 22 79 GCC-\CTCGAGATfAGGGCCGGG Tlus
CPD3 24 73 AAAACTCGAGATGACTAGGCGAATG Tlus
CPD4 22 74 A TICCTCGAGACAGACAGTGCAG,-\ Tlus
CPD5 23 74 CTATCTCGAGCTGCATr�CTGAGC Tlus
CYC P 1 22 75 ACCAGGATCCTAGCTGTCACCG This
CYCP2 24 74 CAGACTGC-\GTITGCGGGTAAATG This
1:'P1 24 74 GTIGCTCGr\GATGTIAACAC-\CCC Tlus
l.'P2 25 77 C-\CCCTCGAGCTGGGA-\ TIGACCAA Tlus
r\OX1 23 76 GTYGCYGGYGTICCIGGL-\ TGGT This
AOX2 23 70 GARG CYT AY A:\ YGARiviG L-\ TGCA Tlus
,-\OX3 23 7 1 GCYTCYTCYTCL-\RRT.-\ICCL\C Tlus studv
AOX4 23 74 TCICKRTGCTIIGCYTCRTCIGC Tlus
AtucL-\OX1 20 65 GTGGTJ"CATGCGCTIAATGG Tlus
AtudAOX2 1 8 60 CTICATGGTCCGGTIACC Tlus
Ben.-\1 2 1 64 GCACTCCGATGAGACTITCTG Tlus
BenA2 21 66 AACATCTGCTGGGTCr�CTCG Tlus
Lacz1 1 8 64 TCGTG,-\CTGGGAAAr\CCC Tlus
Lacz2 1 8 59 CCGTCTGA,\_ TITGACCTG Tlus
Lacz3 1 8 65 ACGGCACGCTGATIGAAG Tlus
Lacz4 1 8 59 CGCTGG.-\ TC,\_,,\_A TCTGTC Tlus
Lacz5 1 8 64 CGG:-\r\GCr\r\r\r\Cr\CCAGC Tlus
Lacz6 1 8 64 CAC-\GKfGTGGA TIGGCG Tlus study
Lacz7 1 8 62 CTGGGATCTGCCATfGTC This
UI6 25 76 CAGGAATAGGATIGCTCGTGCTGAG �IcGiynn
pUC/M 1 3 22 70 GCCAGGGTTITCCCAGTCACGA Perkin Elmer
F01ward
pUC/t £ 1 3 24 70 G,-\GCGG,-\ TA..-\Cr'..-\ TITC-\CACAGG Perkin Elmer Reverse
*Tm CC) was calculated by the formula: 2 x (A+ T) + 4 x (G+C)
Key to symbols: R=A + G, Y=C+T, M=A +C, K=G +T, != Inosine.
Chapter 2
2.8.3 PURIFICATION OF PCR PRODUCTS
Products generated by PCR and required for cloning were purified using a QIAGEN PCR Purification Column Kit, according to the manufacturers instructions. This kit utilises spin cartridges which contain a silica gel membrane that specifically binds DNA and removes impurities.
2.9 CLONING
DNA fragments to be cloned were generated by enzymatic digestion (section 2.7.1) or PCR (section 2.8) , and purified by gel extraction (section 2.7.4) . When necessary, phosphatase treatment (section 2.9 . 1 ) was carried out to prevent re-ligation of the vector fragment, prior to the ligation reaction (section 2.9.2) . The ligation mixes were then transformed by electroporation (section 2.1 0.1) into competent E. coli cells (section 2 . 1 0 . 1 ) .
2.9.1 PHOSPHATASE TREATMENT OF VECTORS
Vector DNA (2-5 !lg) was digested with the appropriate enzyme (section 2.7. 1 ) and the cleaved fragment checked by electrophoresis (section 2.7.2) . The sample was purified by phenol/ chloroform extraction (section 2.6) prior to precipitation by ethanol/isopropanol, after which the pellet was resuspended in 35 IlL of sterile dH20. Approximately 20 ng of vector was then set aside as a 'before treatment' control; the rest of the digested vector underwent phosphatase treatment. The phosphatase used was shrimp alkaline phosphatase
(Invitrogen) and treatment was carried out according to the manufacturers instructions.
2.9.2 DNA LIGATION
GENERAL LIGATION REACTI ONS
In a 1 .5 mL microcentrifuge tube, a reaction mixture was set up containing 2 IlL of 1 0x ligation buffer, x ng of insert DNA, 20 ng of vector DNA (SAP treated) (section 2.9.1) and sterile dH20 to a final volume of 1 9 IlL A 3 IlL 'before ligation' sample was removed prior to addition of 40 U of T4-Ligase (Invitrogen) . The samples were incubated at 4°C overnight. Gel electrophoresis was carried out the following day, with a comparison made
Chapter 2
between a 3 IlL sample of the 'after ligation' mixture, and a 3 IlL 'before ligation' sample.
The following equation was used to estimate the amount of insert required:
Amount of vector x Size of insert
Size of vector (kb)
Amount of insert (ng)
The ratio of vector: insert could be increased from 1 : 1 (eg. 1 :2, 1 :4), thereby enhancing the possibility of a successful outcome.
LIGATIONS WITH THE pGEM SYSTEM
For ligations involving the pGEM vector, the procedure was carried out according to the manufacturers instructions (Promega) .
2.10 TRANSFORMATION OF GENETIC MATERIAL INTO CELLS
Unless otherwise stated, all centrifugation steps for the transformation procedures were carried out at 4°( with 1 5/30
mL
Corex tubes, using a Sorvall centrifuge with a SS34/GSA rotor. Care was taken throughout to ensure that all solutions and equipment used were sterile and ice-cold. A Shimadzu UV - 1 60A UV -visible recording spectrophotometer was used for the determination of cell densities.2.10.1 BACTERIAL TRANSFORMATION
The protocol was obtained from Dower et al (1 988) . All centrifugation steps were carried out with a GSA rotor and 1 00 ml plastic centrifuge tubes.
PREPARATION OF COMPETENT CELLS
An overnight 1 0
mL
culture of the appropriate E. coli strain (XL-1 , Table 2. 1 ) was used to inoculate 1 L of liquid LB (appropriately supplemented) . The culture was grown at 37°C with shaking at 250 rpm for approximately 3 hours (until the cells had reached mid-Chapter 2
exponential phase, with an OD60o of
0.5 - 1 .0) .
The cells were placed on ice for 20 minutes, then harvested by centrifugation at5 000
rpm for1 0 minutes. The cells were
washed sequentially with the following solutions (with subsequent centrifugation at 5 000 rpm for1 0 minutes a fter each washing, and resuspension in the next solution):
1 L of sterile dHzO;0.5
L of sterile dHzO; 20 mL of 1 0% glycerol. The cells were finally resuspended in 4 mL of 1 0% glycerol, aliquoted into 200 )lL volumes and stored at -80°( until required. Immediately prior to electroporation, aliquots of the cells were thawed quickly by gentle flicking of the tube and the tube was then placed on ice.ELECTROPORATION OF COMPETENT CELLS
A
40
).11 aliquot of electroporation competent E. coli cells and the D A sample(2 -4
)lL) were placed in a 1 . 5 mL microcentrifuge tube, gently mL""<ed, and the sample tl1en transferred to an electroporation cuvette (Biorad). A Biorad gene pulser, set at25
)lF, 2.5
kV and 200 0, was used to electroporate the sample, witl1 a time constant of
4 - 5
msec indicating a successful outcome. The cells were inunediately transferred to a1 .5
mL microcentrifuge tube,0.2
mL of liquid LB was added, and tl1e sample then incubated at 3 7° C for1
hour, with shaking at200
rpm. Appropriate dilutions of tl1e rnixture were spread onto LB plates (appropriately supplemented) and incubated at 3 7°( overnight.2.10.2 ASPERGILLUS T RANSFORMATION
Transformation of A. nidulans strains was carried out by PEG pree1p1tauon, with competent cells (protoplasts) prepared by the incubation of mycelia in a hydrolytic solution (Fincham, 1 989) . From liquid media, protoplasts were prepared by tl1e metl1od of Vollmer & Yanofsky (1 986), as modified by ltoh et a/
(1 994).
All centrifugation steps were carried out with a Sorvall centrifuge and a SS34 rotor.PREPARATION OF FUNGAL PROTOPLASTS FROM LIQUID CULTURE
A 1 L flask containing 1 00 mL of liquid MYG (section 2.3.2) (supplemented appropriately) was inoculated with
1
x1 06
spores/mL. The flask was then incubated at 37°C, shaking at250
rpm, for approxin1ately 1 8 hours. The mycelia were harvested by filtration tluough sterile Mira cloth (Calbiochem), and washed thoroughly with sterile dHzO and OM BufferChapter 2
(1 .2 M MgS04.?H20 and 1 0 mM Na2HP04. The pH was adjusted to S.8 by addition of
stock NaH2P04.2H20). The mycelia were then placed in a sterile glass petri dish with
approximately 20 mL of ftlter-sterilised (0.4S J.lM filters, Gelman Sciences) Glucanex
solution (Novo Nordisk) (10 mg/mL, prepared in OM Buffer) and incubated at 37°C for 2
- 2.S hours with gentle shaking.
PREPARATION OF FUNGAL PROTOPLASTS FROM SOLID CULTURE