Filters with 1jj\ of the single stranded and ^|J\ of the double stranded reaction were washed 6 times (5 mins per wash) with 0.5M Na2HP0 4 and twice in water (for 1 min) and dried under a lamp. These filters, together with their
unwashed controls, were Cherenkov counted and comparison of the measurements enabled an estimate of the amount of DNA synthesised in the reactions. The following calculations were used:
3 = Cherenkov constant
15/;ci of 3 zpdCTp lapei were used and tSjUCi = 20nMoles dCTP There are 2.22 X 10® cpm/^ci so If z is the decay factor then
15 X 2.2 X 106 X z X 3 = y cpm/nM dCTP
The washed and unwashed filters from the first strand synthesis were counted and compared.
The control unwashed filter had A cpm
and the washed filter had the lower count, B cpm. B cpm are counts in 1^1
B X total volume X Cherenkov factor (3) = total cpm/reaction The total counts were used to determine the moles of dNTPs
present.
B X total volume X Cherenkov factor = nMoles dNTP/single
Ycpm/nM strand DNA
nMoles/single strand X 350 X 4 X 2 = ng
(350 = molecular weight of a base, multiplied by 4 since there are 4 bases and multiplied by 2 because there are 2 reactions) The total counts/20nMoles were calculated from the unwashed filter and the actual counts added were compared to the
calculated counts added, which helped to indicate how likely the estimated incorporation was. the calculation was as fo llo w s :
15/701 = 20nMoles 3 2pdCTP.
cpm from unwashed filter X total volume X Cherenkov constant = cpm/20nMoles 3 zpdCTP
Estimation of the amount of double stranded cDNA
synthesised in the second strand synthesis reaction was done in exactly the same way except that the final figure was multiplied by 2 to account for the second strand.
These calculations were taken into account when
estimating how much cDNA was to be used in the cloning steps of the library making procedure.
LIGATION OF EcoRI ADAPTORS TO cDNA
EcoRI adaptors were added to the newly synthesised cDNA so that the cDNA fragments could be subcloned into EcoRI cut Xgtll arms. The adaptors were in a ratio of 250pM per ^Jg of DNA and were phosphatase treated to limit the amount of adaptor to adaptor ligation. The ligation reaction was set up as follows (reactions 1 and 2, see above, were pooled):
cDNA 7 jj I
LK buffer (in kit) 2 //I adaptors (in kit) 2 /7 1
H2O 7 /7 1
liaase (in kiti_____ _____ 2/71
t o t a l 20/71
The enzyme was added to the side of the tube and
centrifuged into the reaction. The ligation was incubated on ice for 2 hours at 15°C and then overnight at 15°C. The ligation was stopped by addition of 2/71 of 0.25 EDTA. COLUMN PURIFICATION
A sephadex fractionation column (provided with the cloning kit) was washed with 5ml of TE and the ligation mix made up to 40/71 by addition of TE. The cDNA was applied to the column followed by TE and 30 fractions, of approximately 120/71, were collected. The fractions were Cherenkov counted and the data
plotted graphically against fraction number, diagram is representitive of such a graph.
fractions 9 to 15 (library I) The following adaptors (unlabelled so not detected) Free nucleotides cpm Fraction number
large DNA fragments small DNA fragments
The recovery of DNA was calculated as described previously. KINASING OF DNA LIGATED WITH EcoRI ADAPTORS
To enable ligation of the modified cDNA fragments to EcoRI cut Xgtll arms, a terminal phosphate was added to the adapted cDNA using T4 polynucleotide kinase. The reaction was as fo llo w s ; pooled fractions 900/vl LK buffer 9 5 p I T4 polynucleotide kinase (120Lh _______ 15pl total 101 Op I
This reaction was incubated at 37°C for 45 mins. The
kinased sample was then divided between two Eppendorf tubes and phenol/chloroform extracted twice followed by extraction with an equal volume of chloroform/IAA twice. This solvent was then back extracted with water and added to the original aqueous phase. Butanol extractions (using Aristar™ butanol) were then carried out in order to reduce the volume of the cDNA solution. 2.2 volumes of butanol were added to the cDNA
solution (which was divided into several tubes) and then shaken vigorously, this was repeated until the volume of the cDNA solution was reduced by a factor of 5. The concentrated cDNA was ethanol precipitated at -20°C overnight.
LIGATION OF KINASED cDNA TO VECTOR ARMS
The kinased cDNA was centrifuged for 30 mins at 4°C and the supernatant carefully removed, washed with 70% ethanol, briefly vortexed and repelleted by centrifugation at 4°C for 5 mins. Washes were repeated at 37°C to remove salt. After drying for 5 mins in the freeze drier and resuspension in
water, the following ligation reactions were set up (including reactions with different amounts of cDNA and a control with only Xgtll arms (provided in the kit at 0.5^g/pl)):
arms cDNA oDNA Xgtll 2 7 n g 5 4 n g o n ly cDNA 3.0^1 6 .0 /;l - phosphatased Xgtl 1 arms 2 .0 p l 2 .0 p l 2.0/71 LK buffer 1.0^1 1.0/71 1.0/71
T4 DNA liaase 1.0ul 1.0ul I.Oul
t o t a l 10^1 10/71 1 0/7l
The recommendation of the Amersham™ kit is that between 25ng and 120ng which was suitable for most species and the experiments were carried out within these limits. The
ligations were incubated at 15^0 overnight.
IN VITRO PACKAGING OF LIGATIONS
The cDNA inserts were ligated into Xgtll arms and packaged into protein phage heads. The packaging extracts were carefully thawed and two reactions set up as follows: ligation reaction 10^1
extract A ^Q^J\
extract B 15jul
The reactions were incubated in an ice bucket at 20°C for 2 hours. In order to stop the reaction and to preserve the phage heads 470/71 of PSM were added together with 10/71 of
chloroform (to prevent the growth of bacteria). The libraries were titrated and plated as described below.
X g tll-D M D L (UTRN) LIBRARIES: CHARACTERISATION AND SCREENING
X-bacteriophage stocks and libraries were all titrated to determine the number of plaque forming units (pfu) per ml of stock. 200/71 of Y1088 E. coli cells were mixed with 3mI of top LM-agarose and poured over a plate of LM agar (7cm diameter). The phage stock was spotted, in 10/71 aliquots, around the plate as a range of dilutions, between
1 0 - 2 and 10" ^and incubated at 42°C (in order to facilitate lysis) for «5 hours. The titre was estimated by choosing a dilution where it was possible to count the number of pfus. TRANSFECTION OF E .c o //W IT H Xgt 11-BACTERIOPHAGE
A Y1088 culture was grown overnight in LM broth with maltose and ampicillin at 37°C in a shaking incubator (the cells can be kept at 4°C for up to a week or used later the same day). 2ml of cells were centrifuged for 5mins at 6K rpm and room temperature, the supernatant poured off (although not completely drained) and 10^ pfu phage added, gently mixed and incubated at 37°C for 20mins.
The library (or packaged ligation) was plated by mixing the transfected cells with 8ml of top-agarose and immediately pouring this on top of a warm LM-agar plate. The plates were incubated overnight at 420Q
X - g t ll PLAQUE LIFTS
The X -g tl 1 infected plates were placed at 4°C for 1-2 hours to firm up the top agarose before taking filter lifts. Duplicate nitrocellulose filters were then prepared from each plate by placing the first onto the agarose surface for 1.5 mins and the second for 3 mins. Asymmetric orientation marks were made using a needle, to allow the subsequent
identification of positive clones. The filters were processed by firstly floating them (DNA-side up) for 5 mins in denaturing solution (0.5M NaOH, 1.5M TRIS). The filters were then
submerged for 5 mins in 1M Tris pH8.0, followed by
submergtion for 5 mins in neutralising solution (0.5M Tris, 1.5M NaCI pH7.5), and then rinsing twice in 2 X SSC. Lastly, the filters were air-dried, and baked for two hours at 80°C to fix the DNA onto the membrane.