MINA

I am working on Lambda Phage project. AS a start, I should provide Lambda DNA marker/ EcoRI, Lambda/ Hind III and Lambda/ EcoRI+HindIII. I provided the phage from DSMZ the German company. We have transformed the phage in to the E.coli cells. We have got the lysogenic strains. Then we have tried to shift the bacteria into lytic phase and get the phage. 1 time it went properly and we could get good amount of DNA after purification by DEAE column, recommended in Current Protocol. When we digested the DNA with the given restriction enzymes, the pattern of Lambda/EcooRI was slightly different in one band and in the case of digestion with HindIII, we did not have 3 bands comparing to standard lambda/hind III marker. After so many searches, I thought that there might be something wrong with the host so I should change the host and choose one strain with negative methylation. But in the repeatation I came across we lot of problems. We could never repeat the experiment again and we have no more any phage. We have been involved in doing the last step of the experiment since last 3 months. We have done as the protocols recommend. But we still have some problems. When we provide the lysate, we load it in 3 ways besides to standard DNA phage as follows: Lane 1: Standard DNA phage Lane 2: Direct Lysate Lane 3: Lysate+ DNase+ Dye (including SDS, EDTA) (According to Molecular cloning protocol, Sambrook) Lane 4: Lysate+SDS2%+ EDTA (According to the Current Protocol). For the lane containing DNase we don't see any band. However, it is supposed to see Phage DNA as DNase should have digested the bacterial DNA and SDS and EDTA should decompose the capsid of phage . So, we should see a band in equal size with standard phage DNA. As the Current Protocol describes we should not see any band in the case we load direct lysate and it sounds to be logic as the Phage DNA is inside the capsid and could not be exposed. However, the same reference says that we should see the right band in the case we treat the lysate with SDS and EDTA. Although, we have bands with the right size in both of the cases and in the same intensity. It shows that the phage DNA in our lysate is naked. When we use the DEAE-cellulose for its purification, we have nothing in elutant and we think that it gets bond to the beads due to being naked. Or when we want to extract the DNA by using DNase and the miniprep preparation protocol, the phage DNA gets digested by DNase. I have been very confused. I don’t know if 1. the problem is with the phage or with our techniques. 2. But even when we got good amount of the DNA something wrong with the restriction pattern. 3. I don’t know why our phage is becoming naked or if it is not naked why we have such a pattern after treatment with DNase(Maniatis) or SDS+EDTA (Current Protocol). 4. I don’t know if there is a chance that the propagation of phage is going in the wrong way when it is shifted from the lysogenic phase to lytic one. The most important point is that I am behind the schedule and have lost the phage. I would appreciate your kind attention to give us some advices that how it happens that we have the naked DNA of the phage . Thank you in advance, Mina

Dear Wonder, Could you please read my question / comment on Lambda phage. I think your advices might be helpful for me. Mina

Hi, could you please kindly read my comment and question on Lambda phage. I think you might give me helpful advices. Mina

I am working on Lambda Phage project. . AS a start, I should provide Lambda DNA marker/ EcoRI, Lambda/ Hind III and Lambda/ EcoRI+HindIII. I provided the phage from DSMZ the German company. We have transformed the phage in to the E.coli cells. We have got the lysogenic strains. Then we have tried to shift the bacteria into lytic phase and get the phage. 1 time it went properly and we could get good amount of DNA after purification by DEAE column, recommended in Current Protocol. When we digested the DNA with the given restriction enzymes, the pattern of Lambda/EcooRI was slightly different in one band and in the case of digestion with HindIII, we did not have 3 bands comparing to standard lambda/hind III marker. After so many searches, I thought that there might be something wrong with the host so I should change the host and choose one strain with negative methylation. But in the repeatation I came across we lot of problems. We could never repeat the experiment again and we have no more any phage. We have been involved in doing the last step of the experiment since last 3 months. We have done as the protocols recommend. But we still have some problems. When we provide the lysate, we load it in 3 ways besides to standard DNA phage as follows: Lane 1: Standard DNA phage Lane 2: Direct Lysate Lane 3: Lysate+ DNase+ Dye (including SDS, EDTA) (According to Molecular cloning protocol, Sambrook) Lane 4: Lysate+SDS2%+ EDTA (According to the Current Protocol). For the lane containing DNase we don't see any band. However, it is supposed to see Phage DNA as DNase should have digested the bacterial DNA and SDS and EDTA should decompose the capsid of phage . So, we should see a band in equal size with standard phage DNA. As the Current Protocol describes we should not see any band in the case we load direct lysate and it sounds to be logic as the Phage DNA is inside the capsid and could not be exposed. However, the same reference says that we should see the right band in the case we treat the lysate with SDS and EDTA. Although, we have bands with the right size in both of the cases and in the same intensity. It shows that the phage DNA in our lysate is naked. When we use the DEAE-cellulose for its purification, we have nothing in elutant and we think that it gets bond to the beads due to being naked. Or when we want to extract the DNA by using DNase and the miniprep preparation protocol, the phage DNA gets digested by DNase. I have been very confused. I don’t know if 1. the problem is with the phage or with our techniques. 2. But even when we got good amount of the DNA something wrong with the restriction pattern. 3. I don’t know why our phage is becoming naked or if it is not naked why we have such a pattern after treatment with DNase(Maniatis) or SDS+EDTA (Current Protocol). 4. I don’t know if there is a chance that the propagation of phage is going in the wrong way when it is shifted from the lysogenic phase to lytic one. The most important point is that I am behind the schedule and have lost the phage. I would appreciate your kind attention to give us some advices that how it happens that we have the naked DNA of the phage . Thank you in advance, Mina