Difference between revisions of "Template:Heidelberg/notebook/cf/week33"

Durchführung

Conditions:

After PCR DNA was purified by ethanol precipitation and stored at -20°C

Conditions:

After PCR DNA was purified by Qiagen PCR Purification Kit ans stored at -20°C.

For every reaction:

• Reaction was incubated for 3 h at 37 °C
• After 1.5 h another 2 µL T7 RNA Polymerase were added
• Addition of 2 µL DNase I and further incubation at 37 °C for 20 min

• The constructs containing hammerhead- or HDV-ribozymes was heaten up to 95° for 5 min, so that there is a coplete cleavage.

RNA purification by precipitation:

• Samples was mixed with 200 µL of 2 x loading dye and purified over a 10 % PAGE
• Bands were visualized by UV shadowing and suitable bands were excised
• RNA was eluted out of the gel using 0.3 M NaAc pH 5.5 in three elution steps
• Gel parts were filtered of and 2.5 volumes of -20 °C EtOH were added, sample was stored at -20 °C oN to let the RNA precipitate
• Spin sample at 16,000 g for 30 min, discard the supernatant
• Washed the pellet twice with 70 % EtOH and dissolved RNA in 20 µL of MQ water

• Reaction was incubated for 2 h at 37 °C

• Heat inactivation at 65°C for 10min
• Purification by ethanol precipitation

• Reaction was heaten up to 95°C for 30s without T4 DNA ligase and then cooled down to room temperature
• After 15min  T4 DNA ligase was added.
• Reaction was incubated for 1 h at 37 °C
• Heat inactivation at 80°C for 10min

Procedures:

Digestion of pSB1C3 + MCS +pcat with and without ribozymes:

Description

Reaction mix:

1 µg of DNA

0,1 µl of BamHI-HF

0,1 µl of SalI-HF

2 µl Cutsmart

ad 20 µl ddH2O

Digestion for 1 hour at 37°C

After the reaction the samples were given on a 0,8% Agarose gel and were then purified by gel extraction.

Ligation of the ribozymal fragments into p413

Description:

Chemicals:

1 µl of Vector DNA (about 25 ng) dephosporylated

5 µl of Insert DNA (about 10 ng)

2 µl T4-Ligation Buffer

1 µl T4-Ligase

10 µl ddH2O

The Mix was incubated for 30 minutes at room temperature

Transformation of p413 + Ribozymes + Inserts:

Description:

Steps:

1. Take 50µl chemical competent E. coli from -80 freezer and thaw on ice

1. Add (as master mix):

                2,5µl DNA

                10µl KCM 5x

                37,5µl H2O

1. Incubate on ice for 30 minutes

1. Heat shock at 42°C for 1 minute

1. Incubate on ice for 2 minutes

1. Add 900 µl of LB or 2x YT Medium

1. Incubate on 37°C for 60min

1. Centrifuge 5min at 1000g

1. Take 900µl of supernatant and throw away

1. Resuspend pellet in remaining media

1. Plate out on agar with antibiotics (1:1 / 1:10)

The prior ligations were used for the transformation.

Colony PCR of ribozymes + Inserts:

3 Colonies were picked from every plate which showed 3 or more colonies.

Colonies in the plates after transformation:

Results:

Figure 8: Colony PCR row 1

Figure 9: Colony PCR row 2

Figure 10: Colony PCR row 3

Figure 11: Colony PCR row 4

Figure 13: Colony PCR row 5

Figure 14: Colony PCR row 6

Figure 14: Colony PCR row 7

To check if the azide-modified NTPs are incorporated the reactive azide is clicked to an alkyne activated FAM-alkyne fluorophore under copper catalyzed 1,3-dipolar azide-alkyne cycloaddition (CuAAC). Samples are after the reaction separated on a 20 % denaturing PAGE and visualized first in the fluorescent channel for FAM and afterwards stained with SYBR

Gold to visualize the RNA. Controls were performed as well.

Reaction conditions:

Conditions as Winz, 2012.

• All compounds were mixed in the given order
• Cu(II)SO4 and THPTA and H2O were mixed before adding to the mixture to let THPTA chelat the Cu
• Lastly sodium ascorbate was added to reduce the Cu(II) to Cu(I)
• Reaction was incubated for 1 h at 37 °C and afterwards put at -20 °C

Checking success on a 20 % denaturing PAGE

• Samples were mixed with an appropriate amount of 2 x loading dye and separated on a 20 % denaturing PAGE
• Gel was scanned with a Biorad ChemDoc, GE in fluorescent mode using the pre-set FAM parameters
• Afterwards gel was stained using SYBR gold and scanned with the Typoon in SYBR Gold mode

Result and Outlook:

Labeling of RNA with azide and splinted ligation was only partially successful. Labeling and splinted ligation was repeated with different conditions.