I have transformed agro-bacterium, and I need to amplify the introduced gene (Aska) using PCR without extracting the DNA, I have the bacteria sample as both freezed at -80C in glycerol stock and streaked on LB media plates, I need to know the detailed and exact steps including the reagents and buffers preparations, and what should I consider to avoid any source of error
thanks in advance
Hi there,
Colony PCR is described for Agrobacterium. If it's not working properly with intact cells you may have a go with boiled cell suspension as a template.
Hi there,
Colony PCR is described for Agrobacterium. If it's not working properly with intact cells you may have a go with boiled cell suspension as a template.
- Re suspend your colony in 100ul of 0.1M NaOH
- Subject to heating @ 90C for 15min
- Neutralise with 10ul of 10mM Tris pH 8.0
- Leave over night in the fridge
- Now take 1ul of lysate and perform PCR
I agree with Dominique, colony PCR works well. You just need to heat your template (cell-suspension) longer (5 mins).
Hi,
Below are the steps for preparing template easily for colony PCR:
- Pick a single colony into 10ul sterile water(milliq/millipore) and mix well.(try to streak a plate with same tip/loop for future use).
- heat the tube for 10 min at 99 C.
- Spin the tube 2-3 min at high speed. the supernatant will be you template for PCR.
Hi,
I think you want to confirm your Agrtransformants, that is why you need to amplify some gene of interest. In our lab, we do not go for any screening after Agrobacterium transformation, we directly proceed for the infiltration process as we are confident enough due to the presence of 2 selection markers (specifically Rif & Kan); and it works! So you can take a chance by choosing any isolated colony.
Otherwise, you can proceed with Colony PCR by picking 8-10 colonies from the transformed plate. While suspending the colony into the PCR tube, try to squeeze it using the tip and make master plate using the same tip. During PCR, set the initial step i.e. Denaturation step at 95C for 8-10 minutes. Rest of the things you can proceed as normal PCR. It will work!!!
Directly to the PCR
The PicoPure ™ DNA Kit allows simple and fast extraction of genomic DNA ready for use in PCR. Extract and amplify the DNA in the same tube, without organic extraction phase or column centrifugation. Recover the DNA from some cells obtained by Laser Microdissection or a few milligrams of tissue.
The PicoPure ™ DNA Extraction Kit contains stabilized proteinase K, a reconstitution buffer compatible with PCR techniques, and a complete instructions for use. In this kit, each proteinase K aliquot can be reconstituted freshly to make 150μl of extraction solution, ie 150x10μl using CapSure ™ HS or 30x50μl using Macro CapSure.
Reproducible extraction and amplification of single-copy gene DNA from 10 cells captured by LCM. Microdissected cells from cytospin of white cells of human peripheral blood. Extraction with 10 μl of PicoPure DNA. Analysis after 35 cycles of PCR using 3 sets of primers. M: DNA Marker (Sigma). Corridors 1-5: 536 bp fragment of the human beta-globin gene. Corridors 6-10: 245 bp Cystic Fibrosis Gene Fragment (CFTR). Corridors 11-15: 125 bp fragment of the p53 gene. Corridor 16: no template, PCR negative control.
Increase DNA yield
Since the DNA is not lost during a possible phase of organic extraction or column centrifugation, the PicoPure DNA kit allows a reliable and reproducible DNA yield from a few cells.
PCR studies illustrate the benefits of the PicoPure DNA Kit. An equivalent amount of DNA was transferred to a PCR tube, from extractions made with PicoPure DNA, and with a kit from a leading manufacturer in this market. Real-time quantitative PCR (QRT-PCR) shows that PicoPure DNA processed samples grow faster, suggesting more amount of starting DNA, with more accurate amplification, indicating fewer variations in samples, 'with a classical column technique.
The PicoPure DNA kit provides reliable DNA recovery that increases detection sensitivity, for example, when amplifying a single copy gene in mutation analysis or genotyping assays. This high yield of DNA makes this kit the ideal product for use from small microdissected samples, such as those prepared by LCM.
Superior DNA recovery using PicoPure DNA. 1 μg of DNA was diluted in 10 μl of PicoPure DNA, 5 replicates. The same operation is carried out with other commercial kits. A fragment of the human beta-globin gene is then amplified by RT-PCR, 40 cycles.
For very specific mutations or genotyping assays, the DNA can be extracted from very small samples and directly amplified, and the fragments can be separated by dHPLC (denaturing High-Performance Liquid Chromatography), using a WAVE® system.
I usually perform colony screening by PCR using a protocol similar to that described by Naga. It works very well.
The Naga protocol is good, but the one using the PicoPure DNA kit allows a reliable recovery of the DNA which increases the sensitivity. PicoPure DNA-treated samples grew faster, suggesting more amount of starting DNA, with more faithful amplification, indicating less variation in samples, than with a conventional column technique. Thank you for sharing experience and this is what is changing science.
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