Difference between revisions of "Part:BBa K4436666"

Latest revision as of 14:26, 12 October 2022

Reverse Primer for Lambda Red CsgA knockout

This is the reverse primer for the Lambda Red Knockout of the native csgA in E. Coli.

Usage and Biology

Lambda Red Knockout is a system used by many molecular biologists to knockout single genes or even entire operons (Datsenko and Wanner, 2000). It uses a phage recombinase to replace the native gene in a strain of E. Coli with an antibiotic resistance gene (Datsenko and Wanner, 2000). One of the first few steps of a Lambda Red Knockout is the PCR amplification of a custom primer, with homology to both the antibiotic resistance gene (in the recombinase sites next to it) and the native gene (Datsenko and Wanner, 2000). Accordingly, this primer can be used to amplify the antibiotic resistance gene while allowing for the recombinase to recognize this site and replace the native gene with said gene (Datsenko and Wanner, 2000).

Characterization

We began by preparing a PCR mix on ice, which consisted of:

32.5 microliters of Water

10 microliters of Q5 Reaction Buffer

2.5 Microliters of the Forward Primer

2.5 Microliters of the Reverse Primer

1 microliter of 0.1ng/microliter PKD3

0.5 Microliters of Q5 DNA Polymerase.

We then ran the following PCR Program:

1) Initial Denaturation: 98 C for 30 seconds

2) 30 cycles of PCR

  ->  98 C for 10 seconds
  ->  66 C for 30s
  ->  66 C for 90 s

3) Final Extension: 72 C at 120s

4) Hold at 4 C

Finally, we then ran the results on a 1% Agarose Gel to confirm the results. We were expecting a band at 1.1 kB, which was about the size of the pKD3 plasmid.

As you can see below, we obtained those results around the first week in August. The faint band is in the correct location, and while a primer dimer did form (the band at around 120 bp), the PCR was still sucessful.

References

Datsenko, Kirill A., and Barry L. Wanner. “One-Step Inactivation of Chromosomal Genes in Escherichia Coli K-12 Using PCR Products.” Proceedings of the National Academy of Sciences, vol. 97, no. 12, 2000, pp. 6640–6645., https://doi.org/10.1073/pnas.120163297.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 12: / Line 12: @@
 ===Characterization ===
-We begin by preparing a PCR mix on ice, which consisted of:
+We began by preparing a PCR mix on ice, which consisted of:
 .5 microliters of Water
 microliters of Q5 Reaction Buffer
 .5 Microliters of the Forward Primer
 .5 Microliters of the Reverse Primer
 microliter of 0.1ng/microliter PKD3
 .5 Microliters of Q5 DNA Polymerase.
 We then ran the following PCR Program:
-Initial Denaturation: 98 C for 30 d
+) Initial Denaturation: 98 C for 30 seconds
-cycles
+) 30 cycles of PCR
-C for 10 seconds
+   ->  98 C for 10 seconds
+   ->  66 C for 30s
+   ->  66 C for 90 s
-C for 30s
+) Final Extension: 72 C at 120s
-C for 90 s
+) Hold at 4 C
-Final Extension: 72 C at 120s
-Hold at 4 C
 Finally, we then ran the results on a 1% Agarose Gel to confirm the results. We were expecting a band at 1.1 kB, which was about the size of the pKD3 plasmid.
-As you can see below, we obtained those results around the first month of August.
+As you can see below, we obtained those results around the first week in August. The faint band is in the correct location, and while a primer dimer did form (the band at around 120 bp), the PCR was still sucessful.
+[[File:PKD3 PCR 8-4 gel.png]]