Template:Heidelberg/notebook/pc/week32

week number 32

▼2015-08-04 Colony PCR with ccdBg and ccdBk

5µl One Tag

2x 0.5 µl primer

4 µl water

 

Program:

4min 95°C

30 sec 95°C

30 sec 55°C

2min 68°C

3min 68°C

 

 

A product with 784bp is expected when the ccdB is in p415

Positive colonies were grown for a test digest.

Entryvector test digest with BsaI

600ng p415

0.5 µl BsaI

 

Results:

If the entryvector assembled correctly bands with a side of 4854bp, 2050bp, 577bp and 123bp were expected. Entryvector 1, 2, 4, 5, 6 and 7 can be used.

▼2015-08-04 KCM Trafo

KCM Trafo with correct entryvectors in DH5α to check if it is functional. The original ccdB pSB1C3 plasmid was also transformed as a control.

Results:
With colonies which grow a miniprep and BsaI test digest were done.
 

▼2015-08-06 PCR ribozyme fragments

10µl 5xbuffer
2µl 50mM MgCl2
2x2.5 µl primer
5µl 10mM dNTP
1µl DNA 0.5 µl Velocity
26.5µl water

Program:
97°C 2min
97°C 30sec
61°C 30sec
72°C 45°C
72°C 5min
 

▼2015-08-07 PCR PCR ribozyme fragments optimised

0µl MgCl2

Phusion	Velocity
	5µl 5x buffer
	2x 1.25µl primer
	2.5 µl dNTP
0.5 µl DNA	0.5 DNA
2x 1.25µl primer	0,25 Velocity
9.5 µl water	14.25 water

 

1.5µl MgCl2

Phusion	Velocity
	5µl 5x buffer
	2x 1.25µl primer
	2.5 µl dNTP
0.5 µl DNA	0.5 DNA
2x 1.25µl primer	0,25 Velocity
8 µl water	12.75 water
1.5 µl MgCl2	1.5µl MgCl2

 

 

From some samples a gel extraction was done.