Materials and Method Restriction: 2.

5ug each of pGL2-Basic(200ng/ul), and pJPmdm2 (200ng/ul) plasmids were restricted with Nhe1 and Xba1 respectively in a 25ul reaction setup. Reactions were incubated at 37C for 90 minutes after which the pGL2 Basic vector was treated with 2ul of CIP enzyme for 5 minutes at 37C. Samples of restricted plasmids were run on a 1% low melt agarose; the mdm2 enhancer-promoter fragment was excised form the gel, melted, and a (unpurified) sample used to set up a ligation with the digested Pgl2-basic vector. Ligation: 2ul of the Nhe1 digested and CIP treated pGL2-Basic vector was added to 4ul of the mdm2 enhancer-promoter fragment, with 5ul of 10mMATP, 4ul (5000 units/ml) T4 ligase, and brought to atotal volume of 50ul with sterile water.The following 50ul controlreactions were also set up:digested pGL2-Basic (not CIP treated) with no mdm2 fragment or ligase added; digested pGL2-Basic (not CIP treated) with ligase but no MDM2 fragment; digested vector (CIP treated) with no ligase or MDM2 added; digested (CIP treated) pGL2-Basic with ligase but no mdm2 fragment. For control reactions, 2ul each of vector was used. All reactions contained 5ul of (10mM) ATP, and where applicable, 4ul ofligase was used. Water was added to bring total volumes to 50ul.Reactions were incubated at 16 degrees overnight. Transformation:50ul each the ligation reactions as well as TE buffer(negative control), and native PGL2-Basic(20ug/ml) was added to 200ul each of competent E.coli cells, incubated on ice for 30 minutes, and heat-shocked at 42C for 60 seconds. 1ml of LB broth (no ampicillin) was added to heat-shocked cells and incubated at 37C for 90 minutes. After the 90-minute incubation, 1/10, and 1/100 dilutions of the cells incubated with the native pGL2-Basic, were plated on LB/amp plates along with the remainder of the (undiluted) transformation samples. A sample of the transformationreaction with TE buffer (no DNA, negative control) was plated on no drug plates. Plates were incubated at 37C for 16 hours (overnight). PGL2-Basic-mdm2 (enhancer-promoter segment) Purification and gel electrophoresis: 5ml each of E.coli culture from 4 different clones (obtained from instructor) containingpGl2-Basicmdm2was used in a miniprep plasmid purification after samples were streaked on LB/AMP plates. Cultures were spun for 20 minutes in a clinical centrifuge at max speed. Pellets were resuspended in 1ml each of dilution salts, and pelleted again by spinning in a microcentrifuge at

13000rpm. The pellet from this step was resuspended in 250ul each of resuspension buffer (with RNAse). 250ul each of 0.2N NaoH, 1%SDS was added and mixed to lyse cells. Making sure lysis did not exceed 5 minutes, 350 ul each of High Salt neutralization buffer was added to lysed cells; mixture was centrifuged in microcentrifuge at 13000rpm for 10 minutes. Cleared lysates (supernatant) was added to respective miniprep spin columns and spun at 10000rpm f0r 1 minute. 500ul each of Salt wash buffer was added to columns and spun again at 100000rpm for another minute followed by 750ul of ethanol wash buffer; spun again at 100000 rpm for 1 minute. Columns were spun empty again to rid residual ethanol before eluting DNA with 50ul of TE buffer(10mM Tris Ph7.4, 0.05Mmedta). 5ul reach of the purified plasmids from all 4 clones along with various concentrations (25ug/ml, 50ug/ml,100ug/ml,200ug/ml, 400ug/ml) of native PGL2-Basic were run on 0.8% agarose gel. Restriction analysis: 2.25ugeach of pGL2-Basic, and pJPmdm2, and approximately2.5ug of plasmid DNA isolated from clones 1, 2, 3 and 1.7ug of plasmid isolated form clone 4 were digested with EcoR1in a 25-ul reaction setup. All reactions contained 2.5ul of 10X High Salt buffer. 1ul of EcoR1 enzyme was used per reaction.Samples were incubated at 37C for 90 minutes. After 90-minute incubation, sampleswere run on 1% agarose along with undigested pGL2-Basic, and pJPmdm2 as controls. pGL2-Basic-mdm2 (backward orientation) purification by midiprep: A single clone of pGL2Basic-mdm2(enhancer-promoter segment in backward orientation) was inoculated into 25ml of LB/Amp(100ug/ml) and incubated overnight at 37C, and with a klett reading of 250 was used in the midiprep. Cell culture was pelleted by spinning for 20 minutes in a clinical centrifuge. Pellet was resuspended in 2ml of resuspension buffer (with RNAse). Cells were lysed by adding 2ml of 0.2N NaOH/1%SDS and gently mixing. Lysis was neutralized in less than 5 minutes time elapse by adding 2ml of Neutralization buffer, and the mixture transferred into a Quiafilter and allowed to sit for 10 minutes. 2ml of buffer BB was added to the cleared, and flltered lysate, and applied to a Qiagen plasmid plus Midi column with vaccum suction. 700ul of buffer ETR, was applied to columns, followed by 700ul of buffer PE. Columns were spun in a microcentrifuge at 10000rpm for a minute to remove residual PE buffer. Plasmids were eluted with 200ul of buffer TE. The concentraitions of the purified plasmids were measured with TKO fluorometer. Dilutions (1:2, 1:4, 1:8, 1:16, and 1:32) of the purified PGL2-Basicmdm2(backward) plasmids along with various concentrations(12.5ug/ml, 25ug/ml, 50ug/ml, 100ug/ml, 200ug/ml, and 400ug/ml ) of the native pGL2-Basic was run on a 0.8% agarose gel and later analyzed for expectedband sizes.

RESULTS

pGL2-Basic and pJPmdm2 were succcessfully cut with Nhe1 and Xba1 respectively. The starting point into the Cis-trans experiment was to first excise the mdm2 enhancer and promoter region from pJPmdm2 and ligates it into the pGL2-Basic vector. Figure 1 shows that the mdm2 enhancer and promoter fragment was cut out successfully (compare lanes 7 and 8 with lane 10). Comparing bands in lanes 3 and 5 (pGL2-Basic /Nhe1) with that of lane 1(native pGL2-Basic (lane 1) it could be seen that while the native pGL2-Basic plasmid yielded two bands corresponding to the relaxed and supercoiled forms. The lanes(3, 5) containing the pGL2Basic/Nhe1 had single bands; this is what is expected of a linear vector.

Table 1. Restriction of pJPmdm2 and pGL2-Basic Restriction type PGL2Basic(control) PGL2-Basic/Nhe1 Results No digestion

Complete digestion pJPmdm2(control) No digestion pJPmdm2/Xba1 Complete digestion

Figure 1.Shows Xba1 pJPmdm2 andNhe1 restricted pGL2-Basic along with corresponding native plasmids as controls. Lanes 2, 4, 6, and 9 are empty.Lane1 (PGL2-Basic native), Lane3 (Pgl2-Basic, Nhe1, CIP treated), Lane 5(Pgl2-Basic/Nhe1 non-CIP treated), Lane 7(pJPmdm2/Xba1), Lane 8(pJPmdm2/Xba1), Lane 10(pJPmdm2 native).

Miniprep

Clone 1

Clone2

Clone3

Clone4

EcoR1 fragment 1029 1029 1029 1029 size(bp) Table 3.Sizes of restriction fragment from all four pGL2-Basic-mdm2 (enhancer and promoter) clones

Transformation with the pGL2-Basic-mdm2 (enhancer and promoter) segment was unsuccessful. The mdm2 enhancer promoter segment excised from pJPmdm2 plasmid was ligated into the pGL2-Basic restricted with Nhe1, and the ligation product used to transform competent E. coli. As shown in table 1, with the exception of cells transformed with the native pGL2-Basic, no other sample including the ligation product (T7) yielded transformants. Cells incubated with TE buffer only (T1) did grow on no drug plates but did not grow on AMP plates confirming that the cells were viable prior to transformation.

Tube Dilution #ml # plate d T1 0.1 T1 T2 T3,T 4T5, T6, T7 0.1 0.1 0.1 0.2

Type of plate No drug ampicillin ampicillin ampicillin ampicillin

# of colonies confluen t 0 26 1 0

Transforma nt/ml 0 0 2.6x10^3 1x10^3 0

Average Total transformants transformants /ml 0 0 0 1.8 x10^3 0 0 2.3x10^3 0

10^-1 10^-2

Results from the transformation of competent E.Coli with pGL2-Basic-mdm2 (promoter +enhancer) along with various controls. All reactions tubes contained 200ul of competentcells in addition to the particular transforming reagent. The transformation sample added to a given tube of competent cells are indicated in brackets: Tube T1( TEbuffer, no DNA + Ligase); T2 (native pGL2-Basic, no mdm2 fragment), T3 (non CIP-treated PGL2-Basic, no mdmd2 fragment, no ligase ); T4 (Non CIP treated pGL2-Basic +T4 Ligase, no mdm2 fragment); T5 (CIP treated vector, no mdm2 fragment, no ligase),;T6 (Treated vector+T4 ligase, no mdm2 fragment); T7 (mdm2 fragment+ T4 Ligase + treated pGL2-Basic).

Since the tranformation with the product of the pGL2-Basic+mdm2 enhancer and promoter segment gave no colonies, cultures of E.coli already known to contain the plasmid were obtained and used in miniprep plasmid isolation. As can be seen in figure1, comparing lanes 2-6 and with lanes 7-10 suggested that the starting cultures contained the pGL2-Basic-mdm2 (promoter and enhancer segment).Lanes 2-6 contained the PGL2-Basic plasmid (with no mdm2 fragment). Since the mdm2 fragment is only 400 bp, it was expected that the plasmids isolated from the miniprep cultures if they were positive for the pGL2-Basic+mdme enhancer and promoter segment, should run slightly higher on the gel than the PGL2-Basic only. The

(miniprep plasmid) bands in Lanes 7-10, did not run clearly above the pGL2-Basic only bands however. While figure 1 suggested that the miniprep plasmids may be that of the expected pGL2-Basic mdm2 fragment (lanes 7-10), restriction analysis confirmed that that was the case. The results from the restriction with EcoR1 (figure 2) showed the expected cut-out DNA fragment (1029bp ) when pGL2-Basic+mdm2 enhancer and promoter fragment has the fragment ligated in the backward orientation. All four clones contained the pGL2-Basic with mdm2 in the backward orientation Table 4.concentrations of purified PGL2-Basic-mdm2 (enhancer + promoter segment) from all four clones Miniprep clones 1 Estimated concentration(ug/ml) Concentration by 172 fluorometer(ug/ml) 2 3 4

130

140

81

Figure 2.image of sample DNA purified form four Pgl2-Basic-mdm2 plamids clones. Lanes1, 11, and 12 are empty. Lane 2 (25ug/mlpGL2-Basic), Lane 3(50ug/ml Pgl2-Basic), Lane 4(100ug/ml pGL2-Basic), Lane5( 200ug/ml Pgl2Basic), Lane6(400ug/ml Pgl2-asic), Lane7(miniprep#1), Lane8(miniprep#2), Lane 9(miniprep#3), Lane 10(miniprep #4)

Figure 3.Shows bands of EcoR1 restricted miniprep(Pgl2-Basic-mdm2) plamsids isolated from four clones as well
as EcoR1 restricted pGL2-Basic and pJPmdm2. Lane1 Uncut Pgl2-Basic, Lanes 2-5(unrestricted miniprep clones 1-4), Lane 6 (uncutpJPmdm2), Lane 7(EcoR1 restricted pGL2-Basic), Lane 8-11(EcoR1 restricted miniprep clone 1-4)

Once the purified pGL-2Basic-mdm2 (enhancer+promoter) was confirmed positive, it was necessary to grow large amounts for use in transfection. Figure 4 shows an image of the samples of the pGL2-Basic mdm2 (enhancer+promoter) on agarose gel. They appear identical to the image in figure 2. This is because both images are of the same plasmid except in figure two the samples were loaded with increasing concentrations from left to right while in figure 4 they were loaded in decreasing concentration in the same direction. This corresponds with the respectively increasing and decreasing band intensities in both figures. The concentration of this transfection plasmid as measured with the TKO fluorometer was 200ug/ml

Figure 4.midiprep of Pgl2-Basic-mdm2 (large fragment). Lane 1(400ug/mlpGL2-Basic), Lane 2(200ug/ml pGl2-Basic), Lane3( 100ug/ml Pgl2-Basic) Lane4 (50ug/ml pGL2-Basic), Lane 5(25ug/ml pGL2-Basic), Lane 6(12.5ug/ml, Pgl2-Basic), Lane7-12 contained respective dilutions( 1:2, 1:4,1:8, 1:16, 1:32) of Pgl2-Basic-mdm2(backward orientation)