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Team:Wageningen UR/Journal/week13
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(Created page with " RNA isolation of potato leaf tissue Reverse transcriptase reaction to obtain cDNA PCR on cDNA using coat protein primers") |
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| + | = week 13: 23 july - 29 july = | ||
| - | + | Not much work was done this week because the team spent four days on going to a three day synthetic biology conference in Munich. We learned a lot there and had a great time! | |
| - | + | == Lab work == | |
| - | PCR on cDNA using coat protein primers | + | ''' CCMV ''' |
| + | |||
| + | ''27th July'' (Hugo) | ||
| + | <br/> | ||
| + | Another PCR repeat of 18-07's PCR. Purified PCR products using Fermentas PCR Purification Kit then ran another PCR reaction (repeat of 19-07). | ||
| + | <br/> | ||
| + | <br/> | ||
| + | |||
| + | ''28th July'' (Jeroen) | ||
| + | * Digested IPTG inducible promotor with RBS brick: Spe1 and Pst1 | ||
| + | * Digested CCMV pJET fragment with Xba1 and Pst1 | ||
| + | * after 30 minutes used a PCR purification for digestion inactivation (Pst1 is not heat inactivatable) | ||
| + | * Ligation 1 hour | ||
| + | * Transformation of DH5alpha cells | ||
| + | |||
| + | ''30th July'' (Jeroen) | ||
| + | * 5 colonies picked for colony PCR per plate | ||
| + | |||
| + | |||
| + | '''PLRV''' | ||
| + | |||
| + | * RNA isolation of potato leaf tissue | ||
| + | |||
| + | * Reverse transcriptase reaction to obtain cDNA | ||
| + | |||
| + | * PCR on cDNA using coat protein primers | ||
| + | |||
| + | |||
| + | The figure below shows the purity of isolated RNA. We isolated RNA of different potato cultivars. | ||
| + | |||
| + | [[File:rna2.jpg|frame|center|''Figure 1: isolated RNA PLRV'']] | ||
| + | |||
| + | [[File:table1.jpg|frame|center|''Figure 2: ...'']] | ||
| + | |||
| + | |||
| + | We performed Reverse Transciptase reactions followed by PCR. We used the Revert AID H Minus Reverse Transcription Kit (Fermentas). | ||
| + | |||
| + | [[File:rt-pcr.jpg|frame|center|''Figure 3: ...'']] | ||
| + | |||
| + | Agarose gel of PCR products. Lanes 1 till 4 show the PLRV Coat Protein (CP) bands with the expected size of 627 bp. Lanes 6 till 9 show the PLRV Coat Protein plus Read Through part (CP_RT) with the expected size of 2154 bp. | ||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | ---- | ||
| + | [[https://2012.igem.org/Team:Wageningen_UR/Journal/week12 previous week]] [[https://2012.igem.org/Team:Wageningen_UR/Journal/week14 next week]] | ||
Latest revision as of 03:26, 27 September 2012
week 13: 23 july - 29 july
Not much work was done this week because the team spent four days on going to a three day synthetic biology conference in Munich. We learned a lot there and had a great time!
Lab work
CCMV
27th July (Hugo)
Another PCR repeat of 18-07's PCR. Purified PCR products using Fermentas PCR Purification Kit then ran another PCR reaction (repeat of 19-07).
28th July (Jeroen)
- Digested IPTG inducible promotor with RBS brick: Spe1 and Pst1
- Digested CCMV pJET fragment with Xba1 and Pst1
- after 30 minutes used a PCR purification for digestion inactivation (Pst1 is not heat inactivatable)
- Ligation 1 hour
- Transformation of DH5alpha cells
30th July (Jeroen)
- 5 colonies picked for colony PCR per plate
PLRV
- RNA isolation of potato leaf tissue
- Reverse transcriptase reaction to obtain cDNA
- PCR on cDNA using coat protein primers
The figure below shows the purity of isolated RNA. We isolated RNA of different potato cultivars.
We performed Reverse Transciptase reactions followed by PCR. We used the Revert AID H Minus Reverse Transcription Kit (Fermentas).
Agarose gel of PCR products. Lanes 1 till 4 show the PLRV Coat Protein (CP) bands with the expected size of 627 bp. Lanes 6 till 9 show the PLRV Coat Protein plus Read Through part (CP_RT) with the expected size of 2154 bp.
