Swathi Kumar

Protocol

1) PEX Reaction

Table 1. PEX-EF-46 and PGM1-46, modified by dABFTP.

V=30 µl ds template dABFTP dTTP dGTP KOD KOD buffer H2O1 EF-46 BF 1.071 1.2 0.6 0.6 0.1 3 23.42 EF-46 BF 1.071 1.2 0.6 0.6 0.1 3 23.43 PGM1-46 BF 1.071 1.2 0.6 0.6 0.1 3 23.44 PGM1-46 BF 1.071 1.2 0.6 0.6 0.1 3 23.4

MM 1 5.28 2.64 2.64 0.44 13.20 103.095 EF-46 BF bez E 1.071 1.2 0.6 0.6 0.1 3 23.5

c1 c2

ds template 28 uM 1 uMdAXTP 1 mM 40 uMdTTP 1 mM 20 uMdGTP 1 mM 20 uM

Method

Step 1: Leave mixture for 1 hour at 60oC

Step 2: Purify using Nucleotide removal kit (Qiagen), dilute in 30 µl EB.

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Measuring DNA concentration of PEX products

Table 2. The average concentration (ng/µL) of DNA in EF-BF, PGM1-BF and the control.

Concentration (ng/µL)Sample no. EF-BF PGM1-BF Control

1 40.4 38.7 222 41.4 38.3

Average 40.9 38.5 22

40.9 ng/µL ÷ 38.5 ng/µL= 1.06 x dilution factor

Volume of solution = 28 µl

1.06 x 28 µl = 29.75 µl of EF-BF is needed to get equal concentration (38.5 ng/µl) of PGM1-BF.

29.75 - 28 = 1.75 µl. Therefore, add 1.75 µl of water to EF-BF.

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Result

-1,0 -0,5-0,02

-0,02

-0,01

-0,01

0,00

0,01

0,01

control without enzyme EF-66 BF PGM1-66 BF

I/A

E/V

Figure 1. Electrochemical curves of the PEX products EF-66 BF, PGM1-66 BF and the control (5.10.2016).

Discussion

The PEX reaction did not work for PGM1-66 BF, but it did work for EF-66 BF; nevertheless the signal for EF-66 BF is quite low. (Figure 1).

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2) PAGE analysis of the PEX products

32 P labelling method

Step 1: Create Master Mix using 30.1 µl H2O, 4 µl of PNK buffer, 0.4 µl of PNK enzyme and 1.5 µl of isotope

Step 2: Add 9 µl of Master Mix separately to individual tubes, then add 1 µl of EF-BF, PGM1 and control

Step 3: Heat samples to 37Oc for 30 mins

Step 4: Open sample lids and increase temperature to 90OC for a few minutes

Step 5: Add 5 µl of loading dye to samples for gel electrophoresis

Gel preparation

Step 1: Add 3.22 ml H2O and 1.5ml 10X TBE to 6.3g urea. Mix contents using magnet and Wise Stir machine

Step 2: Add 5.58 ml AA, 75 µl APS and 7.5 µl TEMED to mixture

Step 3: Pour solution into electrophoresis glass slabs and wait 30 mins for gel to set

Step 4: Take set gel out of mould, wash and place in tank with buffer. Let gel run for 30 mins at 1100 V

Step 5: Insert samples into wells in gel run gel

Step 6: Pour out buffer from gel and place on slab dryer for 2 hrs.

Step 7: Place gel on phosphor storage screen under heavy weight.

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Result

Figure 2. Gel image of PEX products EF-66, PGM1-66 and the control.

Discussion

Compared to the control without enzyme, the bend for EF-66 BF is shifted which means that the primer extension worked, although there is some smear which suggests it did not work properly. For PGM1-66, PEX did not work which corresponds to the results gained from electrochemical measurements (Figure 2).

Control without enzyme

EF-66 BFPGM1-66

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3) p53 binding with DNA

Experiment 1

Table 1. Binding- p53wt- products of PEX reaction labelled with BF and NO2, Ab-DO-1, whole complex binding

Step 1 Step 2 Step 320 µl without DNA Total volume 20 µl

SampleNo.

DNA 1 DNA 2 Protein

Ab 10x BB +10x KCl+10xphos

phin

DO-1 (1300ng/µl)

(500ng)

p53-wt (950

ng/µl) (660 ng)

H2O (µl)

DNA 1 (100 ng

or 50 ng)

DNA 2 (100 ng

or 50 ng)

DynaG (µl)

1 EF-46 BF PGM1-50 NO2

p53-wt DO-1 6 0.384 0.694 9.47 1.725 1.725 12

2 EF-46 NO2 PGM1-50 BF p53-wt DO-1 6 0.384 0.694 9.47 1.725 1.725 12MM 13.2 0.84 1.53 20.8

Each 4.4 16.55 µl in 1 tube

c (p53) 950 ng/µlc (DNA) 29ng/µlO.5 M NaCl 10 µl

Method

Step 1: Incubate for 20 mins on ice

Step 2: Mix the two DNAs before adding to the mixture. Incubate for 30 mins

Step 3: Wash magnetic beads three times with 500 µl; binding buffer (1x). Add the reaction mixture to the beads, vortex

Step 4: Incubate at 10oC (1 min, 950 rpm; 9 min 550 rpm – repeat this three times)

Step 5: Wash three times with 100 µl binding buffer (1x)

Step 6: Add 10 µl 0.5 M NaCl. Incubate for 5 mins at 65oC, 600 rpm, remove to a clean tube.

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Result

EF-66 BF + PGM1-66 NO2

EF-66 NO2+ PGM1-66 BF

-1,0 -0,5-0,02

-0,02

-0,01

-0,01

0,00

0,01

0,01

I/A

E/V

Figure 3. Electrochemical curves showing competition between p53 binding with EF-66 BF + PGM1-66 NO2 and EF-66 NO2 + PGM1-66 BF (26.9.2016).

Discussion

The BF peaks are on the left and the NO2 peaks are on the right (Figure 3). As PGM1-66 is specific, it produces a stronger signal when tagged with both NO2 and BF than EF-66. This is because EF-66 is non-specific and therefore has bound with less DNA than PGM1-66.

Swathi Kumar

Experiment 2

Table 2. Binding- p53wt- products of PEX reaction labelled with BF and NO2, Ab-Bp53-10.1, whole complex binding

Step 1 Step 2 Step 3

20 µl without DNA Total volume 20 µl

SampleNo.

Ratio DNA Protein Ab 10x BB +10x KCl+

10xphosphin

Bp53-10.1

(500ng)

p53-wt (950 ng/µl) (660 ng)

H2O (µl)

DNA (100 ng

or 50 ng)

DynaG (µl)

1 1 EF-46 BF p53-wt Bp53-10.1 6 0.385 0.971 10.76 1.887 122 1 PGM1-46 BF p53-wt Bp53-10.1 6 0.385 0.971 10.76 1.887 12

MM 13.2 0.846 2.14 23.7

Each 4.4 18.11 ul in 1 tube

c (Ab) 1300 ng/µlc (p53) 680 ng/µlc (modif. DNA) 53 ng/µlc (non-modif. DNA) 720 ng/µl0.5 M NaCl 20 µl

Method

Same as Experiment 1, except add 20 µl 0.5 M NaCl in step 6 instead of 10 µl.

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Result

EF-66 BF PGM1-66 BF

-1,0 -0,5 0,0-0,04

-0,03

-0,02

-0,01

0,00

0,01

0,02

I/A

E/V

Figure 4. Electrochemical curves showing p53 binding with EF-66 BF and PGM1-66 BF (3.10.2016).

Discussion

PGM1-66 BF produces a stronger signal than EF-66 because it is specific for the DNA sequence being targeted, whereas EF-66 is non-specific (Figure 4).